Conserved domains of glycosyltransferases

Dmitri Kapitonov and Robert K. Yu1

Department of Biochemistry and Molecular Biophysics, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298-0614, USA

Received on October 12, 1998; revised on March 2, 1999; accepted on March 8, 1999

Glycosyltransferases catalyze the synthesis of glycoconjugates by transferring a properly activated sugar residue to an appropriate acceptor molecule or aglycone for chain initiation and elongation. The acceptor can be a lipid, a protein, a heterocyclic compound, or another carbohydrate residue. A catalytic reaction is believed to involve the recognition of both the donor and acceptor by suitable domains, as well as the catalytic site of the enzyme. To elucidate the structural requirements for substrate recognition and catalytic reactions of glycosyltransferases, we have searched the databases for homologous sequences, identified conserved amino acid residues, and proposed potential domain motifs for these enzymes. Depending on the configuration of the anomeric functional group of the glycosyl donor molecule and of the resulting glycoconjugate, all known glycosyltransferases can be divided into two major types: retaining glycosyltransferases, which transfer sugar residue with the retention of anomeric configuration, and inverting glycosyltransferases, which transfer sugar residue with the inversion of anomeric configuration. One conserved domain of the inverting glycosyltransferases identified in the database is responsible for the recognition of a pyrimidine nucleotide, which is either the UDP or the TDP portion of a donor sugar-nucleotide molecule. This domain is termed 'Nucleotide Recognition Domain 1[beta]," or NRD1[beta], since the type of nucleotide is the only common structure among the sugar donors and acceptors. NRD1[beta] is present in 140 glycosyltransferases. The central portion of the NRD1[beta] domain is very similar to the domain that is present in one family of retaining glycosyltransferases. This family is termed NRD1[alpha] to designate the similarity and stereochemistry of sugar transfer, and it consists of 77 glycosyltransferases identified thus far. In the central portion there is a homologous region for these two families and this region probably has a catalytic function. A third conserved domain is found exclusively in membrane-bound glycosyltransferases and is termed NRD2; this domain is present in 98 glycosyltransferases. All three identified NRDs are present in archaebacterial, eubacterial, viral, and eukaryotic glycosyltransferases. The present article presents the alignment of conserved NRD domains and also presents a brief overview of the analyzed glycosyltransferases which comprise about 65% of all known sugar-nucleotide dependent (Leloir-type) and putative glycosyltransferases in different databases. A potential mechanism for the catalytic reaction is also proposed. This proposed mechanism should facilitate the design of experiments to elucidate the regulatory mechanisms of glycosylation reactions. Amino acid sequence information within the conserved domain may be utilized to design degenerate primers for identifying DNA encoding new glycosyltransferases.

Key words: glycosyltransferase/domain structure/classification/nucleotide-binding domain

Introduction

Glycosyltransferases catalyze the synthesis of glycoconjugates, including glycolipids, glycoproteins, and polysaccharides, by transferring an activated mono- or oligosaccharide residue to an existing acceptor molecule for the initiation or elongation of the carbohydrate chain. Because the glycosylation reaction is highly specific with respect to both the anomeric configuration of the sugar residue and the site of the addition, it is expected that unique domain structures for substrate recognition and nucleotide-sugar binding are located within the enzyme molecule. Indeed, common amino acid sequences have been deduced for homologous binding sites. For example, sialyltransferases have sialylmotifs that may participate in the recognition of the donor substrate, CMP-sialic acid (Paulson and Colley, 1985; Datta and Paulson, 1995; Katsutoshi, 1996). The hexapeptide RDKKND in Gal[alpha]1-3 galactosyltransferase and RDKKNE in GlcNAc[beta]1-4 galactosyltransferase have been suggested as the binding site for UDP-Gal (Joziasse et al., 1985, 1989; Joziasse, 1992). These domains have been extensively reviewed in the literature and therefore are not a subject of this review. During the last few years, the amino acid sequences of a number of glycosyltransferases have been identified using sequence data provided by the complete genomic sequences obtained for such organisms as C.elegans, yeasts, several bacteria, and ongoing human and mouse genome projects. These advances have made it possible to deduce conserved domain structures in glycosyltransferases of diverse specificity.

Our interest in this area stems from our recent systematic analysis of the cDNAs encoding UDP-galactose: ceramide galactosyltransferases or CGT (EC 2.4.1.45), the enzyme responsible for the biosynthesis of galactosylceramide. This enzyme has been suggested to play a critical role in myelin formation (Costantino-Ceccarini and Suzuki, 1975; Costantino-Ceccarini and Poduslo, 1989), signal transduction (Dyer and Benjamins, 1989, 1991; Joshi and Mishra, 1992), viral (Baccetti et al., 1991, 1994; Bhat et al., 1991; Harouse et al., 1991; Yahi et al., 1992, 1995; Brogi et al., 1995; Toniolo et al., 1995) microbial adhesion (Garcia Monco et al., 1992; Khan et al., 1996; Kaneda et al., 1997), and oligodendrocyte development (Mirsky et al., 1980; Bansal and Pfeiffer, 1989). In our previous studies we have described the isolation of human, mouse, chicken, and bovine cDNAs for CGT and also the expression of human CGT cDNA (Kapitonov and Yu, 1997a,b). The CGT cDNA sequence appears to be evolutionarily conserved among higher vertebrates; the similarity of CGT from two different systematic classes of vertebrates exceeds 80% and from two different systematic orders of mammals is about 90%. CGT also has a high degree of sequence similarity with glucuronosyltransferases (UGT) (Schulte and Stoffel, 1993; Stahl et al., 1994). All species studied so far have only one copy of the CGT gene. The deduced amino acid sequence of human CGT reveals that it is a 61 kDa protein of 541 amino acid residues, 21 of which are charged. The protein has a calculated pI of 10.36. It maintains all the conserved features of other vertebrate CGTs: a C-terminal transmembrane hydrophobic domain, an endoplasmic reticulum retention signal, and three N-linked glycosylation sites at positions 83, 338, and 447 (none of these are conserved in glucuronosyltransferases). There are six potential sites for casein kinase 2 (CK2) phosphorylation (positions 85, 104, 234, 368, 408, and 410), two potential PKC phosphorylation sites (positions 100 and 426), and a potential N-terminal myristoylation site at position 16 that could be exposed upon cleavage of the signal peptide. Using the amino acid sequence information obtained from the four animal species, we deduced a consensus sequence of CGT, which we used to analyze the database for conserved domains of other glycosyltransferases. In this review, since we analyzed both known and putative glycosyltransferases in order to provide adequate description, all known glycosyltransferases are described by their proper protein names and organisms, while for putative glycosyltransferases from the database, an accession number immediately follows the particular organism. When several genes are located on the same cosmid, the accession number of the cosmid is followed by a dot and a gene number to indicate potential participation in the same biosynthetic pathway in lower eukaryotes. When one protein is chosen to represent a group of highly homologous enzymes, the name of the protein is followed by the number of the family members.

The NRD1[beta] family of glycosyltransferases

When comparing CGT and UGT to some microbial glycosyltransferases, we identified a region with a high degree of similarity, which we have named 'Nucleotide Recognition Domain type 1[beta]" or NRD1[beta], to account for the inversion of anomeric configuration and the resulting configuration of added sugar. This domain is located close to the C-terminal end before the C-terminal transmembrane domain, between residues 357-396 in CGT and between residues 369-407 in UGT. In both CGT and UGT this domain is encoded by one exon. A much smaller area of sequence similarity located closer to the N-terminus is identified as NRD1[beta] S (S for small). In order to understand the functional significance of the NRD1[beta], we generated a homologous amino acid sequence pattern and searched several DNA databases translated in all 6 frames using the programs FindPattern and TFastA (Wisconsin Package, GCG). We found 140 proteins containing this domain. Some representative sequences are shown in Figure 1 together with the enzyme class (if known), gene or protein name, accession numbers and the organisms. If the genes are grouped together on the same chromosome, the accession number of the cosmid followed by dot and the gene number are given. Colocalization of the genes on the same cosmid may give a clue to their function since genes associated with the same biosynthetic pathway tend to group together in the C.elegans genome. Most of these proteins are putative glycosyltransferases encoded in the open reading frames (ORF) found in the genome projects and detected with programs such as Genefinder that utilize criteria such as codon preference and third position bias. The existence of common domains has been described for UGT (Mackenzie et al., 1997). The evolutionary relationship among all major groups of NRD1[beta] glycosyltransferases is shown in Figure 2. Most of the eukaryotic members of NRD1[beta] family have C-terminal transmembrane domain (with the exception of several putative glycosyltransferases from C.elegans, where the exon encoding such a domain could be missed by the program). The topology of NRD1[beta] glycosyltransferases has been studied extensively for UGTs and CGT. All known UGTs have type I transmembrane topology with N-terminus and catalytic domain inside ER and microsomes (Meech and Mackenzie, 1997). Two different topologies were described for CGT biosynthesis: non-hydroxy fatty acid galactosylceramide is synthesized in the cytosolic leaflet of the Golgi, which would be possible only with type II orientation of CGT, while preferred substrate hydroxy-fatty acid galactosyl ceramide is synthesized in the luminal leaflet of the ER (Burger et al., 1996). Since there is only one CGT (Bosio et al., 1996b; Coetzee et al., 1996) that can synthesize both types of GalCer (Schaeren-Wiemers et al., 1995; van der Bijl et al., 1996) and that resides at the density of ER on a sucrose gradient, type I topology with ER localization is probably more accurate, similar to homologous UGT.


Figure 1. Multiple alignment of the NRD1[beta] family of proteins. All conservatively substituted residues that are present in more then 60% of the glycosyltransferases compared are shown as a NRD1[beta] Motif. Signal peptide (SIG), transmembrane domain (TM), potential endoplasmic reticulum retention signal (RETENTION), all truncated, as well as small NRD1[beta] S domain, and large NRD1[beta] L domain, are indicated in Domains. P* indicates the S/T residue in the middle of NRD1[beta] site that could be potentially phosphorylated by CK2. 1 and 2, residues that can potentially participate in the catalytic reaction (E at the position 2). The group number is followed by the type of glycosyltransferase (if known), then by the gene name (if given), then by organism and by accession number. For the multimember group the number of group members is indicated in brackets at the end. For C.elegans the accession number of a cosmid is followed by the gene number. S.c, Yeast Saccharomyces cerevisiae; C.e., Caenorhabditis elegans, nematoda; M.l., Mycobacterium leprae; M.t., Mycobacterium tuberculosis; St.c., Streptomyces capreolus; Ol, oleandomycin; S.a., Streptomyces antibioticus; Ml, macrolide; S.l., Streptomyces lividans; GlyT, glycosyltransferase; B.s., Bacillus subtilis; Z, zeaxanthin; E.u., Erwinia uredovora; A.o., Amycolatopsis orientalis; RhaT, rhamnosyltransferase; P.a., Pseudomonas aeruginosa; B, baumycin; St.c., Streptomyces C5; S.p., Streptomyces peucetius; DauT, daunosaminetransferse; GlcT, glucosyltransferase; S.t., Salmonella typhimurium; Synech, Synechocystis sp, cyanobacteria; Me.j., Methanococcus jannaschii, archaebacteria; G, glucose; N.t., Nicotiana tabacum; L.esc., Lycopersicon esculentum; F, flavonol; Z.m, Zea mais; Petun., Petunia hybrida; I, indole-3-acetate; A.t., Arabidopsis thaliana; C.eleg., Caenorhabditis elegans; UGT, glucuronosyltransferase; P.platessa, Pleuronectes platessa (plaice); CGT, ceramide galactosyltransferase; E, ecdysteroid; NPV, nuclear polyhedrosis virus. The transmembrane domain and signal peptide are truncated. Motif, amino acids residues occurring in more then 60% of UGT1, UGT2, CGT and NRD1[beta]. The conserved [alpha]-helical portion is underlined. Putative proton donor and nucleophile involved in reaction mechanism are marked as 1 and 2 correspondingly. Numbers on the left column indicate the position of the first amino acid residue of the domain; numbers on the right column indicate the total length of the protein. [Rgr], Total length of the protein. Psgene, possible pseudogene; altern, alternative splice sites have been chosen.


Figure 2. The evolutionary relationship between the NRD1[beta] family members. For the explanation of abbreviations used see the legend for Figure 1. All sequences are grouped in five classes. Members of Class I are underlined, members of Class II are shown in italics, Class III of microbial NRD1[beta] glycosyltransferases is shown dark gray, Class IV of plant NRD1[beta] glycosyltransferases is underlined, Class V of animal glycosyltransferases is shown in bold italic. C.elegans sequences are grouped according to similarity into 25 groups. Note that CGT, UGT1 and UGT2 appear as one group of Class V (shown in black). The corresponding division into classes is shown on the left margin by a dotted line.

A potential Ser/Thr residue phosphorylation site is located in the middle of the NRD1[beta] domain that follows the conserved S/TXXE/D pattern for casein kinase 2 (CK2) phosphorylation (where X can be any amino acid residue other than P). Phosphorylation of this site could potentially regulate enzyme activity.

All NRD1[beta] glycosyltransferases can be divided into five classes based on the degree of sequence similarity. Class I is a group of relatively large proteins (950-1050 amino acid (aa) residues) that are homologous to glycosyltransferases from Saccharomyces cerevisiae and Caenorhabditis elegans (C.elegans). Class II constitutes proteins of intermediate size (about 700 amino acid residues) from C.elegans. All bacterial glycosyltransferases fall into Class III. Plant glycosyltransferases constitute Class IV. Class V consists of the remaining glycosyltransferases from C.elegans, glucuronosyltransferases (UGT), and ceramide galactosyltransferases (CGT). Proteins with high degrees of sequence similarity are found in these groups. NRD1[beta] glycosyltransferases are also found in archaebacteria Methanococcus jannaschii, indicating that these enzymes evolved prior to the separation of archaebacteria, eubacteria, and eukaryotes. Protozoa seldom have more than one NRD1[beta] glycosyltransferase, while metazoa have multiple groups of these enzymes.

The most studied metazoan organism, whose genome sequence was about 70% completed at the time of this analysis is C.elegans (Sulston et al., 1992; Watson et al., 1993; Wilson et al., 1994). C.elegans is a free-living nematode comprised of a total of 959 cells with a well-defined pattern of cell division. Based on total protein similarity, we have divided putative glycosyltransferases identified using sequence data from the C.elegans genome into 25 groups, 8 of which contain three proteins or more. Some of the ORF products have been previously identified as similar to UGT. Our analysis indicates that these proteins are not strictly glucuronosyltransferases, but rather glycosyltransferases that are potentially able to catalyze the transfer of either galactose, glucose, or glucuronic acid. In most of the C.elegans putative glycosyltransferases, the NRD1[beta] domain is encoded by one exon.

Function of the NRD1[beta] family members

As a result of our database analysis, we have discovered a new family of glycosyltransferases which we name as the NRD1[beta] family. Class I NRD1[beta] glycosyltransferases include two proteins of 1198 and 949 amino acid residues from S.cerevisiae (Q06321) and C.elegans (Z71177.2), respectively. Class II NRD1[beta] proteins consist of six putative glycosyltransferases from C.elegans of 687-795 amino acid residues. Class III (microbial glycosyltransferases) NRD1[beta] proteins include zeaxanthin glucosyltransferases from erwinia (gracilicutes) and synechocystis (cyanobacteria) that catalyze the conversion of zeaxanthin (Figure 3A) into zeaxanthin-[beta]-diglucoside using UDP-glucose as a substrate. This is a step in the biosynthesis of carotenoid, which is used for protection against ultraviolet light. All known zeaxanthin glucosyltransferases are compared in Figure 4A, and one member of this group is shown in Figure 2.


Figure 3. Chemical structure of the acceptor molecules of the NRD1[beta] glycosyltransferases.


Figure 4. The evolutionary relationship between the NRD1[beta] family members. (A) The evolutionary relationship between all known zeaxanthin glucosyltransferases. GlcT, Glucosyltransferase. (B) The evolutionary relationship between all known glycosyltransferases (GlyT) from Amycolatopsis orientalis; gtfB,C,D,E, glycosyltransferase genes B, C, D, and E. (C) Evolutionary relationship between mycobacterial glycosyltransferases. (D) Comparison of all known flavonol glucosyltransferases. GlcT, Glucosyltransferase; GlyT, glycosyltransferase; CGT7 Manihot, Manihot esculenta, gene 7. (E) Comparison of all known indole-3-acetate glucosyltransferases. GlcT, Glucosyltransferase; Nicotiana, Nicotiana tabacum; Arabidopsis, Arabidopsis thaliana. (F) Evolutionary relationship between all known glucose glucosyltransferases. GlcT, Glucosyltransferase; GlyT, glycosyltransferase; Solanum, Solanum tuberosum. (G) The evolutionary relationship of all known ecdysteroid glucosyltransferases (GlcT). GlcT, Glucosyltransferase; ACMNPV, Autographa californica nuclear polyhedrosis virus (NPV) (O'Reilly and Miller, 1989); BMNPV, Bombyx mori NPV; CFNPV, Choristoneura fumiferana NPV (Barrett et al., 1995); OP, Orgyia pseudotsugata (Pearson et al., 1993); MB, Mamestra brassicae; BS, Buzura suppressaria; LD, Lymantria dispar Multicapsid NPV (Riegel et al., 1994); SL, Spodoptera littoralis; LCGV, Lacanobia oleracea granulosis virus. All Class III microbial glycosyltransferases are shown in gray, all Class IV plant glycosyltransferases are shown in bold, and Class V glycosyltransferases are shown in light gray.

Oleandomycin glycosyltransferase from Streptomyces antibioticus catalyzes the transfer of a glucosyl moiety from UDP-glucose to the 2[prime]-hydroxyl group of desosamine attached to the oleandomycin (Hernandez et al., 1993) (Figure 3B). Macrolide glycosyltransferase from Streptomyces lividans catalyzes the transfer of a glucose or galactose residue from UDP-glucose or UDP-galactose, respectively, to the 2[prime]-hydroxyl group of desosamine or the mycaminose moiety at the C5 position of the lactone ring of 14-membered macrolide antibiotics, including oleandomycin, lankamycin, chalcomycin, and rosaramicin (Jenkins and Cundliffe, 1991; Cundliffe, 1992). Daunosaminetransferase (gene dnrS) from Streptomyces peucetius catalyzes the transfer of a daunosamine residue from TDP-l-daunosamine to [epsis]-Rhodomycinone (Figure 3C) to produce carminomycin (Madduri and Hutchinson, 1995; Otten et al., 1995; Scotti and Hutchinson, 1996). Baumycin glycosyltransferase (gene dauH) from Streptomyces sp. strain C5 may be involved in conversion of the antitumor antibiotic daunomycin to baumycin A1/A2 by catalyzing the addition of glycosyl residues to the 4[prime] position of daunosamine (Ye et al., 1994; Dickens et al., 1995, 1996; Dickens and Strohl, 1996). Rhamnosyltransferase from Pseudomonas aeruginosa catalyzes the transfer of l-rhamnose residue from thymidine-diphosphorhamnose (TDP-l-rhamnose) to [beta]-hydroxydecanoyl-[beta]-hydroxydecanoate (Figure 3D) to produce rhamnolipid (Ochsner et al., 1994). A group of glycosyltransferases from Amycolatopsis orientalis catalyzes the transfer of glucose or xylose residues to the vancomycin heptapeptide (Figure 3E) (Solenberg et al., 1997). All known members of this group are compared in Figure 4B. A separate group of mycobacterial glycosyltransferases consists of putative enzymes with unknown functions. These proteins with their corresponding database accession number are compared in Figure 4C. There are two more Class III microbial glycosyltransferases from Mycobacteria: (Q49841) from Mycobacterium leprae and (AD000002) from Mycobacterium tuberculosum (Figure 2).

Class IV (plant glycosyltransferases) of the NRD1[beta] proteins is composed of several groups. Flavonol 3-O-glucosyltransferases from plants catalyze the transfer of the glucosyl residue from UDP-glucose to flavonol (Figure 3F) to produce flavonol 3-O-d-glucoside in one of the last steps of anthocyanin biosynthesis. All known flavonol glucosyltransferases are compared in Figure 4D and one member is shown in Figure 2. Our sequence analysis indicates that the glucosyltransferase identified as CGT7 glucose:glucosyltransferase (X77464) and a glycosyltransferase from Solanum molongena are probably flavonol glucosyltransferases. A group of indole glucosyltransferases is responsible for the transfer of a glucose residue from UDP-glucose to the indole-3-acetate moiety of plant growth hormone (Figure 3G). All known indole-3-acetate glucosyltransferases are compared in Figure 4E, and one member is shown in Figure 2. Our analysis indicates that jasmonate-induced glucosyltransferase from Nicotiana tabacum (GenBank AB000623) is probably indole-3-acetate glucosyltransferase.

A group of glucose-glucosyltransferases that catalyzes the transfer of a glucosyl residue from UDP-glucose to glucose is compared in Figure 4F, and one member is shown in Figure 2. Our analysis indicates that a glycosyltransferase from Nicotiana tabacum (U32643) and a glycosyltransferase from Lycopersicum esculentum (X85138) twi gene are glucose glucosyltransferases. Rhamnosyltransferase from petunia transfers rhamnosyl residue to anthocyanin.

In addition to CGT and UGT, there are other glycosyltransferases with known functions that belong to Class V of the NRD1[beta] family. Among them are the baculoviral (nucleopolyhedrovirus) ecdysteroid glucosyltransferases (EGTs) that catalyze the transfer of a glucosyl residue from UDP-glucose to ecdysteroids; the latter are insect molting hormones, e.g., ecdysone (Figure 3H) (O'Reilly and Miller, 1989). The expression of EGTs interferes with normal insect development and blocks molting. All known ecdysteroid glucosyltransferases are combined into one group (Figure 4G) of which one member, ACMNPV, is shown in Figure 2. However, one member of the EGT group conjugates ecdysteroids with galactose rather than glucose (O'Reilly et al., 1992).

Group 9 of C.elegans glycosyltransferases contains two proteins, one with 440 amino acid residues and the other with 435 amino acid residues. Both are encoded on the same cosmid: nine C.elegans (Z78200.8) and nine C.elegans (Z78200.7). UGT is a family of microsomal enzymes that transfer glucuronic acid (GlcA) to bilirubin, phenol, and certain toxic xenobiotics and endogenous steroid compounds. Currently there are two groups of highly homologous enzymes: UGT 1 and UGT 2. The UGT1 group consists of eight members: human bilirubin-specific UGT1A (P22309) and UGT1D (P22310), UGT1B (P36509), UGT1C (P35503), UGT1E (P35504), phenol-specific UGT1F (P19224) (Ritter et al., 1992a,b), rat p-nitrophenol-specific UGT1F (P08430) (Iyanagi et al., 1986), and bilirubin-specific UGT1A (P20720) (Sato et al., 1991). Most of these members are derived as a result of alternative splicing with a common C-terminal domain of 245 amino acid residues. The UGT2 group consists of 16 homologous members that include glucuronidate steroid hormones such as androgen, estradiol, and testosterone. Among the UGT2 group are rat 17-[beta]-hydroxysteroid-specific UGT2B6 (P19488) (Mackenzie, 1990), and UGT2B3 (P08542) (Mackenzie, 1987), 3-hydroxyandrogen-specific UGT2B2 (P08541), UGT2B1 (P09875) (Mackenzie, 1986), monoterpenoid alcohol-specific UGT2B12 (P36511) (Green et al., 1995), odorants-specific olfactorial UGT2A1 (P36510) (Lazard et al., 1990), mouse UGT2B5 (P17717) (Kimura and Owens, 1987), rabbit 4-hydroxybiphenyl-specific UGT2B13 (P36512) and UGT2B14 (P36513), UGT2X (P36514) (Tukey et al., 1993), human estriol-specific UGT2B8 (P2376) (Coffman et al., 1990) and UGT2B11 (P36538), UGT2B10 (P36537) (Jin et al., 1993), UGT2B15 (P54855), hyodeoxycholic acid-specific UGT2B4 (P06133) (Jackson et al., 1987), 3,4-catechol-estrogen-specific UGT2B7 (P16662) (Ritter et al., 1990). None of the Tetrapoda UGT has a potential phosphorylation site in the NRD1[beta] domain, except the UGT from fish species. The CGT group transfers galactose residue to ceramide. Currently rat (Schulte and Stoffel, 1993), mouse (Bosio et al., 1996b; Coetzee et al., 1996), human (Bosio et al., 1996a; Kapitonov and Yu, 1997a), chicken, and bovine (Kapitonov and Yu, 1997c) CGTs are cloned. All members of UGT and CGT families are compared in Figure 5A. Group 10 of C.elegans has only one known protein, which is probably a C.elegans analogue of UGT. Group 11 of the NRD1[beta] proteins contains three proteins of 526-530 amino acid residues. These proteins are compared in Figure 5B.


Figure 5. The evolutionary relationship between UGT superfamily (A), members of Groups 11, 13, 15, 16, 17 of C.elegans glycosyltransferases (B), and between members of the DAGGalT family (C).

Group 12 of the NRD1[beta] proteins consists of two proteins, Z75554.5 with 535 amino acid residues and Z75554.6 with 534 amino acid residues. Splice sites other than those predicted with the Genefinder Program are used in alignment on a coding negative strand for (Z75554.6):

Group 13 of the NRD1[beta] proteins consists of 4 proteins: U97009.3,10,1,11, containing 533 to 534 amino acid residues. All Group 13 members are compared in Figure 5B; 13 C.elegans (U97009.10) has two identical copies resulting from an inverted repeat. For Group 14 of C.elegans (AF016420) splice sites different from those predicted by Genefinder are used. Group 15 of C.elegans consists of four members containing 530-570 amino acid residues and one pseudogene that has a deletion of 336 nucleotides. Comparison of Group 15 members is shown in Figure 5B.

Group 16 of C.elegans NRD1[beta] proteins consists of four members with 520 to 542 amino acid residues. All members are compared in Figure 5B. Group 17 consists of seven members with 520-536 amino acid residues, two of which are pseudogenes. Comparison of Group 17 members is shown in Figure 5B. Group 17 C.elegans (U39851.6) is a pseudogene with a point deletion resulting in a frame shift at the amino acid position of 261. In 17 C.elegans (U88311.6) the frame shift is caused by insertion of one nucleotide at the position corresponding to the amino acid residue 268. For Group 17 C.elegans (U97003.6), splicing sites different from those predicted by Genefinder are used. Group 19 consists of two proteins: 19 C.elegans (Z34802.1) with 531 amino acid residues and (AF016424) with 508 amino acid residues.

The NRD1[beta] family of glycosyltransferases encompasses enzymes from eukaryotes, prokaryotes and archaebacteria. Thus, the NRD1[beta] domain probably appeared prior to the separation of these three kingdoms. The acceptor substrates of these glycosyltransferases are either lipids, carbohydrates, proteins, or polycyclic compounds such as steroids, antibiotics, and pigments. These aglycones show little structural similarity (Figure 3). The donor substrates, however, are similar in their nucleotide portion. Therefore, we propose that this domain plays a role in recognition of the nucleotide portion of the sugar-nucleotide substrates, when the nucleotide portion is either UDP or TDP, and the sugar portion is either glucose, galactose, glucuronic acid, xylose, rhamnose, or daunosamine. Occasionally, close members of one group can utilize a different sugar residue, as exemplified by the utilization of galactose by one of the ecdysteroid glucosyltransferases. Potential results of the activity of the NRD1[beta] glycosyltransferases include inactivation of biologically active compounds, as seen with ecdysone, indole acetate, and antibiotics, or the synthesis of new biologically active compounds. All of the enzymes in the NRD1[beta] family probably share a common reaction mechanism, a possibility that could be verified by x-ray crystallographic and/or enzyme kinetic studies.

All C.elegans NRD1[beta] members in the database were detected with the help of the Genefinder Program. It should be noted that this program does make occasional mistakes in selecting the correct splice sites due probably to the lack of information about codon preferences and the third nucleotide bias in C.elegans. The program cannot discriminate between genes and pseudogenes. Occasionally, alternative splicing sites and alternative transcription initiation sites were used based on homology with close NRD1[beta] members. Apart from the complete genes, we found four examples of exon shuffling in the C.elegans genome, where the NRD1[beta]-encoding exon was relocated to other parts of the genome and had undergone several mutations. Gene duplications, deletions, frame shifts and exon shuffling have evolutionary significance in terms of the generation of new genes by recombining domains, point mutations, insertions, and deletions.

After we finished our initial analysis (Kapitonov and Yu, 1997a,b), a similar domain was described (Mackenzie et al., 1997), where the nomenclature for a UDP-glucuronosyltransferase gene superfamily was proposed. Our classification differs from theirs in the following aspects. (1) The NRD1[beta] family of glycosyltransferases is able to utilize not only a UDP-sugar as a donor molecule, but also a TDP-sugar. Therefore, rather than naming these enzymes as UDP glycosyltransferases, we identified them as NRD1[beta]-type glycosyltransferases in order to indicate the presence of a nucleotide recognition domain that can recognize either UDP or TDP. (2) We divide the NRD1[beta] proteins into the following five major classes based on protein sequence homology (Figure 2): Class III of microbial NRD1[beta] glycosyltransferases, Class IV of plant NRD1[beta] glycosyltransferases, animal (including both vertebrate and invertebrate animals) NRD1[beta] glycosyltransferases of typical (440-570 amino acid residues, Class V), medium (about 700 amino acid residues, class II), and large (>800 amino acid residues, Class I) sizes. According to our analysis, some of the genes used in the proposed nomenclature (Mackenzie et al., 1997) are pseudogenes or just the NRD1[beta] exons moved to other parts of the genome; therefore, the assignment of a particular number to those sequences may lead to multiple reassignments later on. Also, for many gene products we used splice sites and initiation codons different from those generated by Genefinder; this resulted in a different proposed evolutionary relationship of the members analyzed.

All C.elegans NRD1[beta] family members have a C-terminal transmembrane domain and C-terminal motif similar to the endoplasmic reticulum retention signal. They can potentially transfer carbohydrate residues onto lipids, carbohydrates, oligopeptides, and polycyclic compounds such as steroid hormones. They also can take part in the biosynthesis of extracellular matrices, hormones, or certain extracellular signal factors. These factors may play an important role in the early stages of multicellular organism development.

The NRD1[alpha] family of glycosyltransferases

CGT can transfer a galactose residue from UDP-galactose to diacylglycerol (DAG) and produce monogalactosyldiacylglycerol (Schaeren-Wiemers et al., 1995). The same function is performed in plants by DAG galactosyltransferase (DAGGalT) (EC 2.4.1.46, 522 amino acid residues), which produces the major structural lipid of chloroplast (Shimojima et al., 1997). DAGGalT shares homology with the murG gene from E.coli and Bacillus subtilis, whose product N-acetylglucosaminyltransferase (GlcNAcT) catalyzes the transfer of N-acetylglucosamine to N-acetylmuramyl-(pentapeptide) phosphoryl-undecaprenol in the last step of peptidoglycan synthesis (Mengin-Lecreulx et al., 1991). This homology implies that the chloroplast membrane biosynthesis mechanism is evolutionarily derived from that of the bacterial cell wall, supporting the endosymbiotic theory (Shimojima et al., 1997). Our computer analysis indicates the existence of two more putative glycosyltransferases from Bacillus subtilis (P54166) and Methanococcus jannaschii (Q58652). These sequences are compared in Figure 5C. The largest area of homology shown in Figure 6 closely resembles that of NRD1[beta].

DAGGalT does not have a terminal transmembrane domain; the first 103 amino acid residues are cleaved upon translocation to the chloroplast. It appears that the murG gene product of E.coli and B.subtilis also lacks an identifiable terminal transmembrane domain although it is membrane associated.

Separation of the NRD1[beta] family members and the NRD1 domain of the DAGGalT family probably occurred prior to the separation of archaebacteria and bacteria; however, we still attribute NRD of the DAGGalT family to NRD1[beta] due to the significant similarity in the central portion of NRD1 domain. The central portion of the NRD1[beta] domain is very close to the conserved domain of a large group of glycosyltransferases. Since all known members of this group are retaining glycosyltransferases, we termed it NRD1[alpha] family to reflect the resulting anomeric configuration. This family is characterized by the presence of two conserved E residues, located at positions 1 and 2 (Figure 6), along with conserved surroundings, spanning about 37 amino acid residues. DAGGalT family is in a way an intermediate family between NRD1[beta] and NRD1[alpha] (closer to NRD1[beta]). Members of this family do not have characteristics for the NRD1[alpha] E residue at position 1 (Figures 1, 6). DAGGalT and B.subtilis (P54166) have K, MurG proteins have R, and M.jannaschii (Q58652) has H at position 1, typical for NRD1[beta]. However, the surrounding regions are not as conserved as for NRD1[beta], where the largest area of homology spans about 60 amino acid residues. Some of the NRD1[alpha] family members are shown in Figure 6. Among them are the family of 17 sucrose synthases, 5 sucrose-phosphate synthases from plants, microbial mannosyltransferases, galactosyltransferases, glucosyltransferases, and galacturonate transferases, as well as several putative glycosyltransferases from C.elegans. All bacterial and some eukaryotic NRD1[alpha] family members apparently lack a transmembrane domain; however, two out of three enzymes from C.elegans (P53993 and Q22698) and one from S.cerevisiae (P53954) have an N-terminal transmembrane domain. None of the above described glycosyltransferases have definable topology.


Figure 6. Members of the NRD1[alpha] family. The type of glycosyltransferase (if known) is followed by the gene name, organism and accession number. ManT, Mannosyltransferase; GalT, galactosyltransferase; GlcT, glucosyltransferase; GalAT, galacturonate transferase; GlyT, glycosyltransferase; GlcNAcT, N-acetylglucosaminyl transferase; CPS, capsular polysaccharide biosynthesis protein; EPS, exopolysaccharide biosynthesis protein; LPS, lipopolysaccharide biosynthesis protein; E.c., Escherichia coli; Vibr.ch., Vibrio cholera; Ye.e, Yersinia enterocolitica; Me.j., archaebacteria Methanococcus jannaschii; S. a., Staphylococcus aureus; St. t., Streptococcus thermophilus; K.p., Klebsiella pneumoniae; Pr. m., Proteus mirabilis; S. p., Streptococcus pneumoniae; C. h., Campylobacter hyoilei; S.e., Salmonella enterica; A.x., Acetobacter xylinum; An, Anabaena sp.; Synech, Synechocustis sp.; S.t, Salmonella typhimurium; Myc. tub., Mycobacterium tuberculosis; Arch. f., Archaeoglobus fulgidus; (17), group of 17 sucrose synthases from both monocotyledonae and dicotyledonae, shown are sucrose synthase from bean and barley; 5, group of five sucrose-phosphate synthases. Shown is sucrose phosphate synthase from rice, TRSD, E, H - LPS biosynthesis proteins; S.c., Saccharomyces cerevisiae; C.elegans, Caenorhabditis elegans; N-GlyT ALG2, asparagine-linked oligosaccharides biosynthesis protein ALG2; B.s., Bacillus subtilis. Motif, amino acid residues present in more then 60% of the family members. NRD1[beta] members MurG protein of Escherichia coli, diacylglycerol (DAG) galactosyltransferase from cucumber, oleandomycin glycosyltransferase, indole-3-acetate glucosyltransferase and CGT Motif are shown for comparison. 1 and 2, amino acid residues that we propose as a part of enzyme reaction mechanism. Residues predicted to be a part of [alpha]-helix in the middle of the proposed catalytic domain are underlined. The three-digit number on the left margin of the alignment indicates position of the first amino acid of the conserved domain; the number on the right margin indicates the total length of the protein.


Figure 7. The evolutionary relationship among members of the NRD1[alpha] family. For the explanation of abbreviations, see the Figure 6 caption. Eukaryotic enzymes are underlined; archaebacterial enzymes are shown in italics.

Function of NRD1[alpha] family members

All of the bacterial glycosyltransferases participate in the formation of the exopolysaccharide (EPS), lipopolysaccharide (LPS), or capsular polysaccharide structures (CPS). Mannosyltransferase C (MtfC) from E.coli is the first ManT that transfers mannose residue to Glc-pyrophosphorylundecaprenol, forming an [alpha](1->3) bond in the O9 antigen biosynthesis pathway. ManT B (MtfB) is the second mannosyltransferase that transfers two mannose residues to form [alpha]-Man-(1->3)-[alpha]-Man-(1->3)-[alpha]-Man-(1->3)-Glc-PP-Undecaprenol intermediate. ManT A (MtfA) transfers three mannose residues to form [alpha]-Man-(1->2)-[alpha]-Man-(1->2)-[alpha]-Man-(1->2)-[alpha]-Man-(1->3)-
[alpha]-Man-(1->3)-[alpha]-Man-(1->3)-Glc-PP-Undecaprenol intermediate. MtfB and MtfA then finish the formation of O9 mannan-repeating unit ->2)-[alpha]-Man-(1->2)-[alpha]-Man-(1->2)-[alpha]-Man-(1->2)-[alpha]-Man-(1->2)-[alpha]-Man-(1-> (Kido et al., 1995). TrsD, E, and H glycosyltransferases participate in the biosynthesis of the O-antigen LPS outer core, serotype O:3 (Skurnik et al., 1995). E.coli K12 GalT is responsible for the formation of an [alpha]-Gal-(1->6) bond in the outer core biosynthesis: [beta]-GlcNAc-(1->6)-[alpha]-GlcIII-(1->2)-[alpha]-GlcII-(1->3)-[-[alpha]-Gal-(1->6)]-[alpha]-GlcI-(1-> inner core (Pradel et al., 1992). CapH of Staphylococcus aureus (Lin et al., 1994; Lee, 1995; Sau and Lee, 1996), Cap1E and Cap1G of Streptococcus pneumonia (Garcia and Lopez, 1997) are glycosyltransferases involved in the biosynthesis of the capsular polysaccharide. Cap1E is a galacturonosyltransferase that catalyzes the formation of an [alpha] (1-3) bond. EpsF and EpsG from Streptococcus thermophilus are involved in the exopolysaccharide (EPS) biosynthesis (Stingele et al., 1996). The family of RfbF galactosyltransferases has three members: GalT from Klebsiella pneumonia serotypes O1 (Q48487; domain starts at aa 277) and O8, and RfbF GalT from Serratia marcescens (Q54481; domain starts at amino acid residue 277). RfbF is capable of forming both [alpha] and [beta] bonds in the LPS core biosynthesis: [beta]-Gal-(1->3)-[alpha]-Gal-(1->3)-[beta]-GlcNAc-(1->lipid (Clarke et al., 1995; Kelly and Whitfield, 1996). CpsF from Proteus vulgaris participates in the formation of capsular polysaccharide (Gygi et al., 1995; Rahman et al., 1997). RfbF from Campylobacter hyoilei is a GalT involved in lipo-oligosaccharide (LOS) biosynthesis (Korolik et al., 1997). RfbP is a GalT that participates in the formation of O:8-antigen repeating unit (Jiang et al., 1991). RfbU is ManT responsible for the synthesis of O-antigen repeating unit of B serogroup: ->2)-[2-O-Ac-[alpha]-Abe-(1->3)]-[alpha]-Man-(1->4)-[alpha]-l-Rha-(1->3)-[[alpha]-Glc-(1->4)]-[beta]-Gal-(1->. RfbW and RfbZ are both ManT responsible for the addition of the second and first mannose to the repeat unit of O-antigen of C2 serogroup correspondingly: ->4-[2-O-Ac-[alpha]-Abe-(1->3)]-[alpha]-l-Rha-(1->2)-[alpha]-Man II-(1->2)-[alpha]-Man I-(1->3)-[alpha]-Gal-(1-> (Brown et al., 1992; Liu et al., 1993; Reeves, 1993; Xiang et al., 1993, 1994). RfbO ManT which catalyzes the transfer of mannose in E1 serogroup O-antigen biosynthesis forming a [beta]-(1->4) bond has no homology within the NRD1[alpha] domain. The remaining enzymes of O-antigen repeat unit biosynthesis RfbV-abequosetransferase and RfbN-rhamnosyltransferase belong to the NRD2 family (see below). AceC from Acetobacter xylinum is a ManT which adds a third carbohydrate residue in the exopolysaccharide acetan biosynthesis (Rha-Glu-Glu-GluA-ManOAc-Glu-Glu)n-PP-Prenol) (Griffin et al., 1994). Not shown are two very closely related ManT from Acetobacter xylinum (Q44571; starts at amino acid residue 272) and GumH from Xanthomonas campestris (Q56774; starts at amino acid residue 279). Among eukaryotes, the same domain is shared by 5 sucrose-phosphate synthases (the one from rice is shown), 17 sucrose synthases from both monocotyledonae and dicotyledonae, 3 putative proteins from C.elegans, and 1 from S.cerevisiae. Not shown in Figure 6 are the following putative glycosyltransferases (accession number is followed by the position of the first amino acid in the conserved domain). Mycobacterium: Q11152 (347) and P54138 (280); Synechocystis: P74348 (270), P74242 (258), P72912 (279), P73402 (312), P73369 (299), P73948 (275), P72902 (301), Q55635 (263), and Q55440 (349); Salmonella: P26388 (308); Bacillus: P71053 (275), P42982 (277), P71055 (283). MurG from E.coli (Mengin-l-ecreulx et al., 1991) as well as some other NRD1[beta] family members are shown for comparison. The evolutionary relationship among the NRD1[alpha]-glycosyltransferases is shown in Figure 7. Note that all eukaryote glycosyltransferases are placed in one group with two subgroups of plant and animal glycosyltransferases according to homology.

Therefore, NRD1[alpha] glycosyltransferases can be further divided into three groups: prokaryotic, eukaryotic plant, and animal glycosyltransferases (very much like NRD1[beta] glycosyltransferases). Apart from the domain shown in Figure 6, some, but not all, retaining glycosyltransferases share an additional domain, where the RXXXXK motif is following a hydrophobic area of five to seven amino acid residues.

The NRD2 family of glycosyltransferases

Human ceramide glucosyltransferase (CGlcT) catalyzes the transfer of the glucosyl residue from UDP-glucose onto ceramide (Ichikawa et al., 1996). In the NRD1[beta] family of enzymes, some members are able to use UDP-glucose as well as UDP-galactose as a donor substrate (e.g., ecdysteroid glycosyltransferase from Spodoptera frugiperda (O'Reilly et al., 1992). Therefore, we attempted to determine whether CGlcT belongs to the NRD1 family. Computer analysis indicated no significant similarity between CGT and CGlcT. Unlike CGT, CGlcT has an N-terminal transmembrane domain. Further analysis indicates that CGlcT belongs to another family of glycosyltransferases that share a common domain, which we have named NRD2. The NRD2 domain is located either immediately adjacent to the N-terminus transmembrane domain, or at the N-terminus. This recognition domain is bipartite: a large NRD2L part of about 50 amino acid residues, located near the N-terminus (amino acid residues 51-93 in the human CGlcT), and a small NRD2S part of 11-17 amino acids (amino acids 136-153 in the human CGlcT). In different NRD2 family members, NRD2L and NRD2S are separated by 27-41 amino acid residues. Human CGlcT has three close homologs, one from Synechocystis and two from C.elegans. Based on the conserved sequence, we have generated a motif and searched the database in order to find additional members of the NRD2 family. We found very similar domains in the NodC protein, cellulose synthase, rhamnosyltransferase, hyaluronate synthase, and some other proteins (Figure 8). All NRD2 family members have a higher degree of similarity within NRD2L than within NRD2S. The evolutionary relationships among the NRD2 family members are shown in Figure 9. Apart from having a common NRD2 domain, all NRD2 family members share other common structures in that they all are membrane-bound proteins and can potentially make contact with the membrane several times as judged by hydropathy plots and turn probabilities. The topology was characterized for CGlcT (Jeckel et al., 1992), DPManT, DPGlcT (Bossuyt and Blanckaert, 1993), and NodC (Barny et al., 1996) enzymes. All of these enzymes have catalytic domains (possibly an NRD2L domain) oriented toward the cytosolic site. The products of these enzymes, such as GalCer, are transported across the membrane either into the Golgi (Lannert et al., 1994) into the ER such as Dol-P-Glc and Dol-P-Man (Schutzbach and Zimmerman, 1992), or outside the cell such as the Nod factors. The N-terminal transmembrane domain may cross the membrane into the extracytoplasmic space. Most of the bacterial members of the NRD2 family lack an N-terminal transmembrane domain, but still have several potential transmembrane domains throughout the enzyme. The existence of homologous enzymes sharing the same topology may provide an evolutionary link between bacterial cell surface membranes and the eukaryotic ER and Golgi apparatus.


Figure 8. The conserved domain of the NRD2 family members. DPManT, dolichol-phosphate mannosyltransferase; S.c., Saccharomyces cerevisiae; DPGlcT, dolichol-phosphate glucosyltransferase; NODC, nodulation protein C; Rh.m., Rhizobium meliloti; FBFA, fruit body formation protein A; St.a., Stigmatella aurantiaca; HASA, hyaluronate synthase; S.py., Streptococcus pyogenes; EXOM, O, U, W, and A, exoolygosaccharide biosynthesis proteins M, O, U, and W (glucosyltransferases) and A; R. m., Rhizobium meliloti; GlcNAcT, N-acetyl glucosaminyltransferase; S.e., Staphylococcus epidermidis, ICAA protein; EPSI and EPSG, exopolycaccharide biosynthesis proteins I and G; S.t., Streptococcus thermophilus; GalT, galactosyltransferase; CPSJ and CPS I, capsid polysaccharide biosynthesis proteins J and I; L.l., Lactococcus lactis; GGAB and GGAA TeicAc, teichoic acid biosynthesis protein B and A; B.s., Bacillus subtilis; S.p., Streptococcus pneumoniae; GlyT KFIC, glycosyltransferase KFIC; E. c., Escherichia coli; GlyNAcT LGTA, N-acetyl glucosaminyl/galactosaminyltransferase LGTA protein; N.m., Neisseria meningitidis; M.j., Methanococcus jannaschii; VIRB V.a., Vibrio anguillarum, VIRB protein; RFBV, B, Q, glycosyltransferases; Sa.t., Salmonella typhimurium; Y.e., Yersinia enterocolitica; RhaT RFBN and RFBQ, rhamnosyltransferase RFBN and Q BM_1_proteins; Sa.t., Salmonella typhimurium; S.d., Shigella dysentariae; EXPE2, exopolysaccharide biosynthesis protein E2; S.m., Sinorhizobium meliloti; ACSA, cellulose synthase; A.x., Acetobacter xylinum; CerGlcT, ceramide glucosyltransferase. NRD2 Motif, amino acid residues that are present in the most of the NRD2 family members. Number in front of the name indicates the belonging to a group of several members. The number of group members is shown in brackets. 28-42aa, Nonconserved region of 28-42 amino acid residues not shown in the figure.


Figure 9. The evolutionary relationship among the NRD2 family members. Eukaryotic proteins are underlined and archaebacterial proteins are shown in italics. Number in front of the name indicates a group of several enzymes that share higher homology with the members of a group, than with other members of NRD2 family. For an explanation of the abbreviations see the Figure 8 caption.

Function of NRD2 family members

Based on sequence similarities, the NRD2 family of glycosyltransferases can be divided into the following groups. Group 1 consists of GDP-mannose: dolichol-phosphate O-[beta]-d-mannosyltransferase (EC 2.4.1.83). This is an essential membrane-bound enzyme responsible for the synthesis of dolichol-phosphate-mannose (DPM), a key glycosyl donor for the synthesis of the N-linked oligosaccharide chains of glycoproteins (Orlean et al., 1988; Orlean, 1990; Schutzbach et al., 1993). Other enzymes of the NRD2 family that belong to this group are DPM from yeast, DPM1 from Trypanosoma brucei (Q26732) and Ustilago maydis (P54856), hypothetical proteins from Synechocystis sp (P74505) and Mycobacterium tuberculosis (P71781), and two proteins from E.coli (P77293 and P77757). Dolichol-phosphate glucosyltransferase from Saccharomyces cerevisiae transfers glucose from UDP-glucose to dolichol phosphate (Heesen et al., 1994). An absence of this enzyme leads to underglycosylation of the secreted proteins.

Group 2 of the NRD2 glycosyltransferase family consists of NODC proteins. These proteins are produced by rhizobial microorganisms that fix nitrogen inside specialized nodule structures in the root. NODC proteins possess N-acetylglucosaminyl transferase activities (chitin synthases) that are involved in the formation of lipo-chito-oligosaccharide Nod factors that initiate root nodule morphogenesis in legumes. NODC proteins are responsible for the synthesis of trimers to pentamers of [beta]-1,4-linked GlcNAc residues called Nod factors (Figure 10A) (Geremia et al., 1994), which are also acylated (Debelle et al., 1996). Further modifications by acetylation or sulfation are achieved at R and R[prime][prime] which determine host specificity (Schultze et al., 1992). Nod factors can be further fucosylated, and one microorganism can produce several Nod factors to broaden the host range (Lerouge et al., 1990; Price et al., 1992). At concentrations of 10-7 to 10-6 M, secreted Nod factors can induce root hair curling and the formation of nodules (Relic et al., 1994; Dazzo et al., 1996). So far nine NODC proteins from different rhizobial strains of the genuses Rhizobium (P04341, P50357, P50356, P17862, P04340, P24151, and P04678) Azorhizobium (Q07755), and Bradirhizobium (P53417 and P04677) have been described (Ueda et al., 1995). All these proteins maintain a conserved NRD2 domain.


Figure 10. Chemical structure of the acceptors of the NRD2 glycosyltransferases.

Another example of cell to cell interaction in prokaryotes can be found in the development of the myxobacterium Stigmatella aurantiaca, which undergoes a multicellular cycle of development resulting in the formation of fruiting bodies. The protein FBFA (Q53680), which participates in the fruiting body formation, is a glycosyltransferase that shares a higher degree of homology with the NODC protein than it does with any other protein of the NRD2 family. Inactivation of FBFA leads to the formation of nonstructured clumps rather than the structured fruiting body (Silakowski et al., 1996). Together with two other putative enzymes from Bacillus subtilis (P96587) and Synechocystis sp (P74165), FBFA forms a third group of NRD2 glycosyltransferases.

Several groups of the NRD2 enzymes participate in cell wall formation. Hyaluronan synthase, or HASA, from group A streptococci (e.g., Streptococcus pyogenes), produces the antiphagocytic EPS hyaluronate capsule by alternating the addition of UDP-N-acetyl-d-glucosamine and UDP-d-glucuronic acid. The enzyme apparently has both glycosyltransferase activities (Dougherty and van de Rijn, 1992; DeAngelis et al., 1993). Streptococcal hyaluronate is chemically indistinguishable from that found in animal connective tissues (Figure 10B).

EXO is a group of glycosyltransferases responsible for EPS type I succinoglycan biosynthesis in Rhizobium meliloti. EPS plays an important role during interaction with its host alfalfa. EXO glycosyltransferases participate in the biosynthesis of the repeating unit of succinoglycan (Figure 10C). EXOM adds the 4th, EXOO the 5th, EXOU the 6th, and EXOW the 7th sugar residue in the succinoglycan chain (Glucksmann et al., 1993; Reuber and Walker, 1993). EXOA, which adds the 2nd sugar, has a high similarity with the NRD2 motif in the NRD2L portion, but a lower similarity in the NRD2S portion. The 8th sugar is modified by addition of pyruvate, the 7th sugar is linked to succinate, and the 3rd is acetylated. The whole structure is anchored to the membrane by a lipid carrier (Reinhold et al., 1994).

The ICAA protein from Staphylococcus epidermidis participates in the synthesis of the polysaccharide intercellular adhesin (PIA), which is located on the cell surface and is responsible for the formation of the biofilm and the large cell clusters. PIA is a linear [beta]-1,6-linked glycosaminoglycan composed of at least 130 2-deoxy-2-amino-d-glucopyranosyl residues, most of which are N-acetylated. ICAA has been shown to be a membrane-bound N-acetylglucosaminyltransferase that forms [beta]-1,6-linked N-acetyl-d-glucosaminyl polymers (Heilmann et al., 1996).

Group 4 of the NRD2 glycosyltransferase family is the group of 13 enzymes responsible for cell surface polysaccharide (CSP) biosynthesis. CSP can be attached to the cell membrane as the O-antigen of lipopolysaccharides (LPS) to form a capsule around the cell as capsular polysaccharides (CPS). It can also be completely excreted as exopolysaccharides (EPS). The biological functions of the polysaccharides are diverse, including roles in pathogenesis and symbiosis, protection of the cell from desiccation or other environmental stresses, and/or facilitating adherence of bacteria to cell surfaces.

The EPSI glycosyltransferase from Streptococcus thermophilus Sfi6 (Q56046) participates in the synthesis of excreted EPS having the repeating unit ->3)-[beta]-d-Galp-(1->3)-[[alpha]-d-Galp-(1->6)]-[beta]-d-Glcp-(1->3)-[alpha]-d-GalpNAc-(1-> which is responsible for the extremely slimy texture of some yogurt products (Stingele et al., 1996). The EPSG glycosyltransferase from Lactococcus lactis (O06035) participates in the synthesis of similar EPS (van Kranenburg et al., 1997).

CPSJ and I proteins of Streptococcus pneumoniae (O07340 and O07339) are glycosyltransferases that transfer the 4th sugar (galactose) and 3rd sugar (N-acetylglucosamine), respectively, to the glycolipid intermediate (L):

during the synthesis of capsular polysaccharides.

Minor teichoic acid synthesis proteins GGAA and GGAB from Bacillus subtilis (P46917 and P46918) are glycosyltransferases that are involved in the synthesis of poly(3-O-[beta]-d-glucopyranosyl N-acetylgalactosamine-1-phosphate), a secondary anionic polymer of gram-positive bacteria (Mauel et al., 1994; Lazarevic et al., 1995). In E.coli KFIC protein (Q47330) is responsible for the synthesis of the capsular polysaccharide, K5 antigen, a polymer of 4)-[beta]-GlcA-( 1,4)-[alpha]GlcNAc-( 1. KFIC can use both UDP-glucuronic acid (UDP-GlcA) and UDP-N-acetyl-glucosamine (UDP-GlcNAc) as substrates (Petit et al., 1995).

Other proteins that belong to Group 4 of the NRD2 glycosyltransferases are Haemophilus influenzae (Q57022 and Q56869) (Fleischmann et al., 1995), Yersinia enterocolitica (Q56869, Q57022), and TRSB protein (Q56914). The latter is responsible for the biosynthesis of LPS O:3 antigen (homopolymer of 6-deoxy-l-altrose repeating units linked by 1,2 linkages) (Skurnik et al., 1995); Bacillus subtilis (P71059 and P71057) (Glaser et al., 1993), E.coli (P11290), Synechocystis sp. spore coat polysaccharide biosynthesis protein SPSA (P73983), and Pseudomonas aeruginosa mucosa-induced protein MIGA (P95448).

Group 5 of the NRD2 glycosyltransferases consists of enzymes involved in lipopolysaccharide (LPS) synthesis. The LGTA glycosyltransferase from Neisseria meningitidis (Q51115) is responsible for the synthesis of meningococcal LPS oligosaccharide of L3 immunotype. It transfers an N-acetylglucosamine residue to the terminal galactose (4th sugar) of the inner core lacto-N-neotetraose (the last four sugars): [beta]-d-Gal-(1->4)-[beta]-d-GlcNAc-(1->3)-[beta]-d-Gal-(1->4)-[beta]-d-Glc-(1->4)-[alpha]-Hep-(1->5)-[alpha]-KDO (Jennings et al., 1995). Its counterpart in Neisseria gonorrhoeaerhea (Q50946) is encoded by the lsi-2 gene (Danaher et al., 1995). Other members of the group 5 glycosyltransferases include putative proteins from Haemophilus influenzae (Q57287), Bacillus subtilis (P71054) and LGTD protein from Neisseria gonorrhoeaerhea (Q50949) and meningitidis (P96946) that transfer GlcNAc to the terminal Gal of the lacto-N-neotetraose portion of the lipo-oligosaccharide (LOS) inner core (Gotschlich, 1994). Q50951 from Neisseria gonorrhoeae is a putative protein. In the fish pathogen Vibrio anguillarum VIRB protein takes part in the synthesis of the LPS, which is a major surface flagella sheath antigen (Norqvist and Wolf-Watz, 1993).

Group 6 of the NRD2 glycosyltransferases includes RFBV, an abequose transferase from Salmonella enterica, serogroup C2 (P26401), which takes part in the formation of the O-antigen repeat unit (Reeves, 1993):

RFBN is a rhamnosyltransferase of Salmonella enterica serogroup B (Q54129) that participates in the formation of O-antigen: ->2)-[beta]-Man-[-(1->3)-[alpha]-2OAcAbe]-(1->4)-[alpha]-l-Rha-(1->3)-[alpha]-Gal-[-(1->4)-[alpha]-Glc]-(1->. The same function is performed in the Salmonella enterica serogroup E1 by the orf11.9 product (Liu et al., 1993). Other enzymes in this group include the product orf14.1 of Salmonella enterica (Q99192), which probably performs the same role as that encoded by orf11.9 of serogroup, two proteins from Mycoplasma genitalium (P47306 and P47271) (Fraser et al., 1995), one protein from Yersinia enterocolitica (Q56866), and two proteins from Synechocystis sp. (P72899 and P73996).

Rhamnosyltransferases RFBQ (Q03581) and RFBR (Q03582) from Shigella dysenteria and RFBF from Shigella flexneri (P37782) belong to Group 7 of the NRD2 family. RFBQ transfers the 2nd sugar residue (Rha I) and RFBR transfers the 3rd sugar residue (Rha II) to the O-antigen repeating unit of the cell surface LPS: ->2)-[alpha]-l-Rha II-(1->3)-[alpha]-l-RhaI-(1->2)-[alpha]-d-Gal-(1->3)-[alpha]-d-GlcNAc-(1->-Acyl Carrier Lipid (ACL). RFBF transfers a Rha III residue in the Y serotype biosynthesis reaction to form O-antigen: ->2)-[alpha]-l-RhaIII-(1->2)-[alpha]-l-RhaII-(1->3)-[alpha]-l-RhaI-(1->3)-[beta]-d-GlcNAc-(1->-ACL (Morona et al., 1995). Other glycosyltransferases in this group are the following: RFBC from Yersinia enterocolitica (S35296) (Zhang et al., 1993) that participates in 6-deoxy-l-altrose (C3 epimer of rhamnose) polymerization, RFBF rhamnosyltransferase from Leptospira interrogans (P71441) (Mitchison et al., 1997), rhamnosyltransferase from Streptococcus pneumoniae (O07868), and RFBE glycosyltransferase from Klebsiella pneumoniae (Q48482) (Kelly and Whitfield, 1996). EXPE2 from Synorhizobium meliloti participates in the formation of EPS II, which consists of alternating glucose and galactose residues joined by [alpha]-1,3 and [beta]-1,3 linkages.

Cellulose synthases from Acetobacter xylinum (P19449 and P21877) (Wong et al., 1990; Standal et al., 1994) and Paramecium bursaria Chlorella virus 1 (U42580) (Kutish et al., 1996) belong to the 8th group of NRD2 glycosyltransferases. These enzymes are responsible for the synthesis and crystallization of cellulose (Figure 10D).

The last and the most distant group of the NRD2 glycosyltransferases is formed by ceramide glucosyltransferases from humans, two putative proteins from C.elegans, and one putative protein from Synechocystis. The existence of two proteins in C.elegans may indicate that these enzymes utilize different substrates, such as hydroxy ceramide and nonhydroxy ceramide.

Similar to members of the NRD1 family, members of the NRD2 family of glycosyltransferases are found in eukaryotes, prokaryotes, bacteria, and archaebacteria, indicating that this domain appeared prior to separation of these three kingdoms. All NRD2 members are membrane-bound enzymes that can potentially span the membrane several times. It is likely that the evolution of this family of enzymes was closely connected with the evolution of the membrane structure. In addition, we have identified a conserved region of these proteins that is located near the N-terminus or immediately following the N-terminal transmembrane domain (if such a domain is present).

Results of our computer analysis allow us to predict that it is possible to identify new members of the membrane-bound NRD2 family of glycosyltransferases through the following strategies: (1) using degenerate primers based on the conserved amino acid sequence of the NRD domains, (2) hybridization with the NRD portion of known genes, or (3) screening expression libraries (e.g., the [lambda]gt11 library with polyclonal antibodies against the NRD2 domain). We can also speculate that glycosyltransferases needed for eukaryotic glycolipid and bacterial LPS biosynthesis evolved from a common predecessor. Therefore, based on the sequence homology with glycosyltransferases involved in the later steps of LPS biosynthesis, one can expect to discover new glycosyltransferase members of the NRD1 or NRD2 family that are membrane-bound and catalyze the addition of sugar residues to glycoconjugates. Since glycoconjugates play a crucial role in embryonic development, future research should be directed toward identifying those glycoconjugates by isolating NRD1 and NRD2 glycosyltransferases from higher eukaryotes and, in particular, mammals, and analyzing their function.

Catalytic domain and proposed reaction mechanisms

When we analyzed the NRD1[alpha] family of glycosyltransferases, we determined that all the members of that family transfer carbohydrate residues with the retention of configuration. Comparison of the NRD1[beta] and NRD1[alpha] glycosyltransferase family members shows that one of the distinctive features of the two families is the presence of two conserved glutamic acid residues (marked with numbers 1 and 2 in Figure 6) with E at position 2 in the middle of a short and mostly uncharged [alpha]-helical domain (about 3 turns). We propose that these E residues may play a functional role as a base in a nucleophilic substitution reaction that proceeds through a double displacement mechanism with an oxocarbenium ion transition state (Davies et al., 1998a,b) (Figure 11A). These E residues would be negatively charged under physiological conditions. E at position 2 is located above the plane of the sugar and serves as a nucleophile, while E at the position 1 is located beneath the plane and serves as a proton donor at the first step of the reaction. The pK difference between D and E side chains (3.96 and 4.32, respectively) may account for the exclusive selection of E as a nucleophile since it would be expected to create a more stable transition bond than D. The hydrophobic environment of a catalytic domain may further increase the pK (up to 8.2 for E; Fersht, 1977). In the case of NRD1[alpha] glycosyltransferases, the donor substrate is always a sugar-nucleotide (either purine or pyrimidine), whereas the acceptor can be a growing carbohydrate chain, lipid, or protein. For simplicity, only a carbohydrate is shown as an acceptor in Figure 11. The family of retaining [beta]-glycoside hydrolases also utilizes two E residues in the catalytic domain with the reaction proceeding in the opposite direction through acid/base catalyzed formation and subsequent hydrolysis of a covalent glycosyl-enzyme intermediate (Davies et al., 1998a).


Figure 11. Proposed reaction mechanisms for the glycosyltransferase reaction with retention of the sugar donor configuration (A), or inversion (B).

All known NRD1[beta] glycosyltransferases transfer sugar residue with the inversion of configuration that would require one nucleophile. We noticed that E residue at position 2 and the [alpha]-helical domain are conserved in the NRD1[beta] (Figure 1) and NRD1[alpha] (Figure 6) families of glycosyltransferases. We propose that this glutamic acid residue can play a role as a single nucleophile in the glycosyltransferase reaction with inversion of configuration. In a single nucleophile mechanism, SN2, nucleophilic substitution will result in an inversion of the anomeric configuration (Figure 11B). At position 1, eight amino acid residues away from the conserved E residue at position 2, is an H residue in the NRD1[beta] family or R/K in DAG GalT. These residues are located at exactly the same distance from the conserved E, and therefore could be located underneath the sugar plane such as E at position 1 in the NRD1[alpha] family. H and R/K may play a role in the donation of a proton (Figure 11B). Another motif that is conserved among all NRD1 family members is the PQ and/or DQ motif (Figure 1). Since all NRD1 glycosyltransferases can accept only pyrimidine-sugar, Q residue may play a role in the recognition of pyrimidine residue (like in EQQN motif; Boeggeman and Qasba, 1998).

Since NRD1[alpha] glycosyltransferases can accept both purine- and pyrimidine-containing sugar nucleotides, they are lacking conserved nucleotide recognition motifs. If our model is correct, substitution of E residue at position 2 for D in any NRD1[beta] or NRD1[alpha] family member should cause a dramatic reduction in the reaction rate, while substitution for any other amino acid should completely eliminate the enzyme activity. Substitution of H residue for R/K at position 1 in NRD1 family members could possibly be tolerated with some changes in the reaction rate. Substitution of H/R/K residues at the position 1 for E may convert inverting glycosyltransferase into retaining and convert resulting anomeric configuration from [beta] to [alpha]. Glycosyltransferases with S/T residues located two amino acid residues before residue E at position 2 could be potentially regulated by phosphorylation since placing a strong negative charge in the catalytic domain between two amino acid residues involved in the reaction should affect enzyme activity. Some microbial NRD1 glycosyltransferases apparently do not have a conserved E residue at position 2 (Figure 1). Since the homology in the surrounding area is high enough to attribute these enzymes to the NRD1[beta] family, we believe that the catalytic domain of these enzymes evolved from the NRD1[beta] catalytic domain to accommodate large molecules such as antibiotics that some of these enzymes inactivate or produce (such as vancomycin (Figure 3E) or baumycin). Therefore these glycosyltransferases utilize alternative nucleophile spaced further away.

The NRD2 family includes inverting glycosyltransferases that can accept both purine- and pyrimidine-containing sugar nucleotides. At this point we are unable to propose any particular amino acid residue as a candidate for the nucleophile or for nucleotide recognition, but the YN...E motif, E at the position 1 and two of the DD motifs (at positions 2 and 3) are good candidates for the beginning of site-directed mutagenesis experiments (Figure 8). Some of the conserved D residues at the position 3 fall into the conserved motif DXD proposed for metal binding (Boeggeman and Qasba, 1998).

Classification of glycosyltransferases and concluding remarks

Based on our analysis, the most logical way to classify the glycosyltransferases that we have analyzed is according to the type of bond they form and the reaction mechanisms they carry out their catalytic function. Thus, all glycosyltransferases analyzed fall into two types: retaining glycosyltransferases that transfer sugar with the retention of configuration of anomeric carbon, and inverting glycosyltransferases that transfer sugar with the inversion of configuration of anomeric carbon. The retaining glycosyltransferases that we have analyzed are characterized by the presence of two E residues in the proposed catalytic domain and are further divided into the class of prokaryotic glycosyltransferases and eukaryotic glycosyltransferases within two subclasses of animal and plant glycosyltransferases. The inverting glycosyltransferases that we have analyzed are divided into 2 subclasses: H/(R/K)-E subclass with H or R/K residue at position 1 and E residue at position 2 and NRD2 subclass. Each of these subtypes is further divided into the class of prokaryotic and eukaryotic glycosyltransferases. The latter is further divided into two subclasses of plant and animal enzymes. The division of NRD1[beta] glycosyltransferases into five classes described in Figure 2 is based on the comparison of the full length proteins. Comparison of the catalytic or entire NRD1[beta] domain will bring all animal NRD1[beta] glycosyltransferases together by eliminating the variation in amino acid chain lengths. The second subtype of inverting glycosyltransferases (with the NRD2 domain) is also divided into prokaryotic and eukaryotic glycosyltransferases. According to recent classification (Campbell et al., 1997) most of NRD1[beta] (H(R/K)-E) domain-containing glycosyltransferases fall into family 1, NRD1[alpha] (E-E) domain-containing glycosyltransferases fall into family 4, NRD2 glycosyltransferases fall into family 2 and 21 of nucleotide-diphospho-sugar glycosyltransferases (Campbell et al., 1997). There are numerous glycosyltransferases without E-E (NRD1[alpha]), H(R/K)-E (NRD1[beta]), or NRD2 domains. Therefore, additional subtypes will be introduced upon accumulation of the data.

In conclusion, we have described glycosyltransferases that belong to three different families according to the similarity and proposed catalytic domain based on the most conserved amino acid residues. Using computer alignment of known glycosyltransferases, we generated patterns that we used to search the database for additional putative glycosyltransferases. Based on the amino acid residues present in the catalytic domain and the type of bond these glycosyltransferases catalyze, we have proposed reaction mechanisms and classified glycosyltransferases. Further studies by site-directed mutagenesis and/or x-ray structure analysis are required for the confirmation of our model. The crystallographic information can be used for the design of enzyme inhibitors. Because some of the NRD1 and NRD2 enzymes are indispensable proteins in bacteria, these inhibitors could lead to a new class of antibiotics. Since microbial glycosyltransferases are much closer to each other than to eukaryotic glycosyltransferases (Figures 2, 7, 9), it is possible to design inhibitors specific only for microorganisms. Additionally, suicide inhibitors can be designed that can be used for modification of NRD1 or NRD2 enzymes in order to purify and characterize new glycosyltransferases. Another way to search for new glycosyltransferases is to use PCR with degenerate primers synthesized based on the amino acid sequence of the conserved domains. The complete listing of all analyzed glycosyltransferases with conserved domains is available on the web site (www.views.vcu.edu/~glyco).

Acknowledgments

This work was supported by a USPHS Grant NS11853-25. We thank Drs. Cara-Lynne Schengrund and H.Tonie Wright for their helpful discussion of the manuscript.

Abbreviations

NRD, nucleotide recognition domain; CGT, ceramide galactosyltransferase; UGT, glucuronosyltransferase; DAG, diacylglycerol; DAGGalT, diacylglycerol galactosyltransferase; GlcNAcT, N-acetylglucosaminyltransferase; GalNAcT, N-acetylgalactosaminyltransferase; EPS, exopolysaccharide; LPS, lipopolysaccharide; CPS, capsular polysaccharide structures; ManT, mannosyltransferase; LOS, lipooligosaccharide; GalT, galactosyltransferase; GlcAT, glucuronosyltransferase; CGlcT, ceramide glucosyltransferase; DPM, dolichol phosphate mannose; DPManT, dolichol-phosphate mannosyltransferase; ER, endoplasmic reticulum.

References

Baccetti ,B., Benedetto,A., Burrini,A.G., Collodel,G., Elia,C., Piomboni,P., Renieri,T., Sensini,C. and Zaccarelli,M. (1991) HIV particles detected in spermatozoa of patients with AIDS. J. Submicrosc. Cytol. Pathol., 23, 339-345. MEDLINE Abstract

Baccetti ,B., Benedetto,A., Burrini,A.G., Collodel,G., Ceccarini,E.C., Crisa,N., Di Caro,A., Estenoz,M., Garbuglia,A.R., Massacesi,A. and et al. (1994) HIV-particles in spermatozoa of patients with AIDS and their transfer into the oocyte. J. Cell Biol., 127, 903-914. MEDLINE Abstract

Bansal ,R. and Pfeiffer,S.E. (1989) Reversible inhibition of oligodendrocyte progenitor differentiation by a monoclonal antibody against surface galactolipids. Proc. Natl. Acad. Sci. USA, 86, 6181-6185. MEDLINE Abstract

Barny ,M.A., Schoonejans,E., Economou,A., Johnston,A.W. and Downie,J.A. (1996) The C-terminal domain of the Rhizobium leguminosarum chitin synthase NodC is important for function and determines the orientation of the N-terminal region in the inner membrane. Mol. Microbiol, 19, 443-453. MEDLINE Abstract

Barrett ,J.W., Krell,P.J. and Arif,B.M. (1995) Characterization, sequencing and phylogeny of the ecdysteroid UDP-glucosyltransferase gene from two distinct nuclear polyhedrosis viruses isolated from Choristoneura fumiferana. J. Gen. Virol., 76, 2447-2456. MEDLINE Abstract

Bhat ,S., Spitalnik,S.L., Gonzalez-Scarano,F. and Silberberg,D.H. (1991) Galactosyl ceramide or a derivative is an essential component of the neural receptor for human immunodeficiency virus type 1 envelope glycoprotein gp120. Proc. Natl. Acad. Sci. USA, 88, 7131-7134. MEDLINE Abstract

Boeggeman ,E. and Qasba,P.K. (1998) Use of site directed mutagenesis to identify the [beta]1-4-galactosyltransferase binding sites for metal ions and UDP-Galactose. Glycobiology, 8, 1139.

Bosio ,A., Binczek,E., Le Beau,M.M., Fernald,A.A. and Stoffel,W. (1996a) The human gene CGT encoding the UDP-galactose ceramide galactosyl transferase (cerebroside synthase): cloning, characterization and assignment to human chromosome, 4, band q26. Genomics, 34, 69-75. MEDLINE Abstract

Bosio ,A., Binczek,E. and Stoffel,W. (1996b) Molecular cloning and characterization of the mouse CGT gene encoding UDP-galactose ceramide-galactosyltransferase (cerebroside synthetase). Genomics, 35, 223-226. MEDLINE Abstract

Bossuyt ,X. and Blanckaert,N. (1993) Topology of nucleotide-sugar:dolichyl phosphate glycosyltransferases involved in the dolichol pathway for protein glycosylation in native rat liver microsomes. Biochem. J., 296, 627-632. MEDLINE Abstract

Brogi ,A., Presentini,R., Piomboni,P., Collodel,G., Strazza,M., Solazzo,D. and Costantino-Ceccarini,E. (1995) Human sperm and spermatogonia express a galactoglycerolipid which interacts with gp120. J. Submicrosc. Cytol. Pathol., 27, 565-571. MEDLINE Abstract

Brown ,P.K., Romana,L.K. and Reeves,P.R. (1992) Molecular analysis of the rfb gene cluster of Salmonella serovar muenchen (strain M67): the genetic basis of the polymorphism between groups C2 and B. Mol. Microbiol, 6, 1385-1394. MEDLINE Abstract

Burger ,K.N., van der Bijl,P. and van Meer,G. (1996) Topology of sphingolipid galactosyltransferases in ER and Golgi: transbilayer movement of monohexosyl sphingolipids is required for higher glycosphingolipid biosynthesis. J. Cell Biol., 133, 15-28. MEDLINE Abstract

Campbell ,J.A., Davies,G.J., Bulone,V. and Henrissat,B. (1997) A classification of nucleotide-diphospho-sugar glycosyltransferases based on amino acid sequence similarities [letter] [published erratum appears in Biochem. J. 1998 Feb 1;329 (Pt 3):719]. Biochem. J., 326, 929-939. MEDLINE Abstract

Clarke ,B.R., Bronner,D., Keenleyside,W.J., Severn,W.B., Richards,J.C. and Whitfield,C. (1995) Role of Rfe and RfbF in the initiation of biosynthesis of d-galactan I, the lipopolysaccharide O antigen from Klebsiella pneumoniae serotype O1. J. Bacteriol., 177, 5411-5418. MEDLINE Abstract

Coetzee ,T., Fujita,N., Dupree,J., Shi,R., Blight,A., Suzuki,K. and Popko,B. (1996) Myelination in the absence of galactocerebroside and sulfatide: normal structure with abnormal function and regional instability. Cell, 86, 209-219. MEDLINE Abstract

Coffman ,B.L., Tephly,T.R., Irshaid,Y.M., Green,M.D., Smith,C., Jackson,M.R., Wooster,R. and Burchell,B. (1990) Characterization and primary sequence of a human hepatic microsomal estriol UDPglucuronosyltransferase. Arch. Biochem. Biophys., 281, 170-175. MEDLINE Abstract

Costantino-Ceccarini ,E. and Poduslo,J.F. (1989) Regulation of UDP-galactose:ceramide galactosyltransferase and UDP-glucose:ceramide glucosyltransferase after crush and transection nerve injury. J. Neurochem, 53, 205-211. MEDLINE Abstract

Costantino-Ceccarini ,E. and Suzuki,K. (1975) Evidence for presence of UDP-galactose:ceramide galactosyltransferase in rat myelin. Brain Res., 93, 358-362. MEDLINE Abstract

Cundliffe ,E. (1992) Glycosylation of macrolide antibiotics in extracts of Streptomyces lividans. Antimicrob. Agents Chemother., 36, 348-352. MEDLINE Abstract

Danaher ,R.J., Levin,J.C., Arking,D., Burch,C.L., Sandlin,R. and Stein,D.C. (1995) Genetic basis of Neisseria gonorrhoeae lipooligosaccharide antigenic variation. J. Bacteriol., 177, 7275-7279. MEDLINE Abstract

Datta ,A.K. and Paulson,J.C. (1995) The sialyltransferase 'sialylmotif" participates in binding the donor substrate CMP-NeuAc. J. Biol. Chem., 270, 1497-500. MEDLINE Abstract

Davies ,G., Mackenzie,L., Varrot,A., Dauter,A., Brzozowski,M., Schulein,M. and Withers,G. (1998a) Snapshots along the enzymatic reaction coordinate: analysis of a retaining [beta]-glycoside hydrolase. Biochemistry, 37, 11707-11713. MEDLINE Abstract

Davies ,G., Sinnott,M. and Withers,S. (1998b) Glycosyl transfer. In Sinnott,M. (ed.), Comprehensive Biological Catalysis, Vol. 1. Academic Press, New York, pp. 119-208.

Dazzo ,F.B., Orgambide,G.G., Philip-Hollingsworth,S., Hollingsworth,R.I., Ninke,K.O. and Salzwedel,J.L. (1996) Modulation of development, growth dynamics, wall crystallinity and infection sites in white clover root hairs by membrane chitolipooligosaccharides from Rhizobium leguminosarum biovar trifolii. J. Bacteriol., 178, 3621-3627. MEDLINE Abstract

DeAngelis ,P.L., Papaconstantinou,J. and Weigel,P.H. (1993) Molecular cloning, identification and sequence of the hyaluronan synthase gene from group A Streptococcus pyogenes. J. Biol. Chem., 268, 19181-19184. MEDLINE Abstract

Dean-Nystrom ,E.A. and Samuel,J.E. (1994) Age-related resistance to 987P fimbria-mediated colonization correlates with specific glycolipid receptors in intestinal mucus in swine. Infect. Immun., 62, 4789-4794. MEDLINE Abstract

Debelle ,F., Plazanet,C., Roche,P., Pujol,C., Savagnac,A., Rosenberg,C., Prome,J.C. and Denarie,J. (1996) The NodA proteins of Rhizobium meliloti and Rhizobium tropici specify the N-acylation of Nod factors by different fatty acids. Mol. Microbiol, 22, 303-314. MEDLINE Abstract

Dickens ,M.L. and Strohl,W.R. (1996) Isolation and characterization of a gene from Streptomyces sp. strain C5 that confers the ability to convert daunomycin to doxorubicin on Streptomyces lividans TK24. J. Bacteriol., 178, 3389-3395. MEDLINE Abstract

Dickens ,M.L., Ye,J. and Strohl,W.R. (1995) Analysis of clustered genes encoding both early and late steps in daunomycin biosynthesis by Streptomyces sp. strain C5. J. Bacteriol., 177, 536-543. MEDLINE Abstract

Dickens ,M.L., Ye,J. and Strohl,W.R. (1996) Cloning, sequencing and analysis of aklaviketone reductase from Streptomyces sp. strain C5. J. Bacteriol., 178, 3384-3388. MEDLINE Abstract

Dougherty ,B.A. and van de Rijn,I. (1992) Molecular characterization of a locus required for hyaluronic acid capsule production in group A streptococci. J. Exp. Med., 175, 1291-1299. MEDLINE Abstract

Dyer ,C.A. and Benjamins,J.A. (1989) Organization of oligodendroglial membrane sheets. II. Galactocerebroside:antibody interactions signal changes in cytoskeleton and myelin basic protein. J. Neurosci. Res., 24, 212-221. MEDLINE Abstract

Dyer ,C.A. and Benjamins,J.A. (1991) Galactocerebroside and sulfatide independently mediate Ca2+ responses in oligodendrocytes. J. Neurosci Res., 30, 699-711. MEDLINE Abstract

Fersht ,A. (1977) Enzyme Structure and Mechanism. W. H. Freeman, New York.

Fleischmann ,R.D., Adams,M.D., White,O., Clayton,R.A., Kirkness,E.F., Kerlavage,A.R., Bult,C.J., Tomb,J.F., Dougherty,B.A., Merrick,J.M., et al. (1995) Whole-genome random sequencing and assembly of Haemophilus influenzae Rd [see comments]. Science, 269, 496-512. MEDLINE Abstract

Fraser ,C.M., Gocayne,J.D., White,O., Adams,M.D., Clayton,R.A., Fleischmann,R.D., Bult,C.J., Kerlavage,A.R., Sutton,G., Kelley,J.M. et al. (1995) The minimal gene complement of Mycoplasma genitalium [see comments]. Science, 270, 397-403. MEDLINE Abstract

Garcia ,E. and Lopez,R. (1997) Molecular biology of the capsular genes of Streptococcus pneumoniae. FEMS Microbiol. Lett., 149, 1-10. MEDLINE Abstract

Garcia Monco ,J.C., Fernandez Villar,B., Rogers,R.C., Szczepanski,A., Wheeler,C.M. and Benach,J.L. (1992) Borrelia burgdorferi and other related spirochetes bind to galactocerebroside. Neurology, 42, 1341-1348. MEDLINE Abstract

Geremia ,R.A., Mergaert,P., Geelen,D., Van Montagu,M. and Holsters,M. (1994) The NodC protein of Azorhizobium caulinodans is an N-acetylglucosaminyltransferase. Proc. Natl. Acad. Sci. USA, 91, 2669-2673. MEDLINE Abstract

Glaser ,P., Kunst,F., Arnaud,M., Coudart,M.P., Gonzales,W., Hullo,M.F., Ionescu,M., Lubochinsky,B., Marcelino,L., Moszer,I. et al. (1993) Bacillus subtilis genome project: cloning and sequencing of the 97 kb region from 325 degrees to 333 degrees. Mol. Microbiol, 10, 371-384. MEDLINE Abstract

Glucksmann ,M.A., Reuber,T.L. and Walker,G.C. (1993) Family of glycosyl transferases needed for the synthesis of succinoglycan by Rhizobium meliloti. J. Bacteriol., 175, 7033-7044. MEDLINE Abstract

Gotschlich ,E.C. (1994) Genetic locus for the biosynthesis of the variable portion of Neisseria gonorrhoeae lipooligosaccharide. J. Exp. Med., 180, 2181-2190. MEDLINE Abstract

Green ,M.D., Clarke,D.J., Oturu,E.M., Styczynski,P.B., Jackson,M.R., Burchell,B. and Tephly,T.R. (1995) Cloning and expression of a rat liver phenobarbital-inducible UDP- glucuronosyltransferase (2B12) with specificity for monoterpenoid alcohols. Arch. Biochem. Biophys., 322, 460-468. MEDLINE Abstract

Griffin ,A.M., Morris,V.J. and Gasson,M.J. (1994) Genetic analysis of the acetan biosynthetic pathway in Acetobacter xylinum. Int. J. Biol. Macromol., 16, 287-289. MEDLINE Abstract

Gygi ,D., Rahman,M.M., Lai,H.C., Carlson,R., Guard-Petter,J. and Hughes,C. (1995) A cell-surface polysaccharide that facilitates rapid population migration by differentiated swarm cells of Proteus mirabilis. Mol. Microbiol., 17, 1167-1175. MEDLINE Abstract

Harouse ,J.M., Bhat,S., Spitalnik,S.L., Laughlin,M., Stefano,K., Silberberg,D.H. and Gonzalez-Scarano,F. (1991) Inhibition of entry of HIV-1 in neural cell lines by antibodies against galactosyl ceramide. Science, 253, 320-323. MEDLINE Abstract

Heesen ,S., Lehle,L., Weissmann,A. and Aebi,M. (1994) Isolation of the ALG5 locus encoding the UDP-glucose:dolichyl-phosphate glucosyltransferase from Saccharomyces cerevisiae. Eur. J. Biochem., 224, 71-79. MEDLINE Abstract

Heilmann ,C., Schweitzer,O., Gerke,C., Vanittanakom,N., Mack,D. and Gotz,F. (1996) Molecular basis of intercellular adhesion in the biofilm-forming Staphylococcus epidermidis. Mol. Microbiol., 20, 1083-1091. MEDLINE Abstract

Hernandez ,C., Olano,C., Mendez,C. and Salas,J.A. (1993) Characterization of a Streptomyces antibioticus gene cluster encoding a glycosyltransferase involved in oleandomycin inactivation. Gene, 134, 139-140. MEDLINE Abstract

Ichikawa ,S., Sakiyama,H., Suzuki,G., Hidari,K.I. and Hirabayashi,Y. (1996). Expression cloning of a cDNA for human ceramide glucosyltransferase that catalyzes the first glycosylation step of glycosphingolipid synthesis. Proc. Natl. Acad. Sci. USA, 93, 4638-4643. MEDLINE Abstract

Iyanagi ,T., Haniu,M., Sogawa,K., Fujii-Kuriyama,Y., Watanabe,S., Shively,J.E. and Anan,K.F. (1986) Cloning and characterization of cDNA encoding 3-methylcholanthrene inducible rat mRNA for UDP-glucuronosyltransferase. J. Biol. Chem., 261, 15607-15614. MEDLINE Abstract

Jackson ,M.R., McCarthy,L.R., Harding,D., Wilson,S., Coughtrie,M.W. and Burchell,B. (1987) Cloning of a human liver microsomal UDP-glucuronosyltransferase cDNA. Biochem. J., 242, 581-588. MEDLINE Abstract

Jeckel ,D., Karrenbauer,A., Burger,K.N., van Meer,G. and Wieland,F. (1992) Glucosylceramide is synthesized at the cytosolic surface of various Golgi subfractions. J. Cell Biol., 117, 259-267. MEDLINE Abstract

Jenkins ,G. and Cundliffe,E. (1991) Cloning and characterization of two genes from Streptomyces lividans that confer inducible resistance to lincomycin and macrolide antibiotics. Gene, 108, 55-62. MEDLINE Abstract

Jennings ,M.P., Bisercic,M., Dunn,K.L., Virji,M., Martin,A., Wilks,K.E., Richards,J.C. and Moxon,E.R. (1995) Cloning and molecular analysis of the Isi1 (rfaF) gene of Neisseria meningitidis which encodes a heptosyl-2-transferase involved in LPS biosynthesis: evaluation of surface exposed carbohydrates in LPS mediated toxicity for human endothelial cells. Microb. Pathog., 19, 391-407. MEDLINE Abstract

Jiang ,X.M., Neal,B., Santiago,F., Lee,S.J., Romana,L.K. and Reeves,P.R. (1991) Structure and sequence of the rfb (O antigen) gene cluster of Salmonella serovar typhimurium (strain LT2). Mol. Microbiol., 5, 695-713. MEDLINE Abstract

Jin ,C.J., Miners,J.O., Lillywhite,K.J. and Mackenzie,P.I. (1993) cDNA cloning and expression of two new members of the human liver UDP-glucuronosyltransferase 2B subfamily. Biochem. Biophys. Res. Commun., 194, 496-503. MEDLINE Abstract

Joshi ,P.G. and Mishra,S. (1992) Galactocerebroside mediates Ca2+ signaling in cultured glioma cells. Brain Res., 597, 108-113. MEDLINE Abstract

Joziasse ,D.H. (1992) Mammalian glycosyltransferases: genomic organization and protein structure. Glycobiology, 2, 271-277. MEDLINE Abstract

Joziasse ,D.H., Bergh,M.L., ter Hart,H.G., Koppen,P.L., Hooghwinkel,G.J. and Van den Eijnden,D.H. (1985) Purification and enzymatic characterization of CMP-sialic acid: [beta]- galactosyl1-3-N-acetylgalactosaminide [alpha]2-3-sialyltransferase from human placenta. J. Biol. Chem., 260, 4941-4951. MEDLINE Abstract

Joziasse ,D.H., Shaper,J.H., Van den Eijnden,D.H., Van Tunen,A.J. and Shaper,N.L. (1989) Bovine alpha 1-3-galactosyltransferase: isolation and characterization of a cDNA clone. Identification of homologous sequences in human genomic DNA. J. Biol. Chem., 264, 14290-14297. MEDLINE Abstract

Kaneda ,K., Masuzawa,T., Yasugami,K., Suzuki,T., Suzuki,Y. and Yanagihara,Y. (1997) Glycosphingolipid-binding protein of Borrelia burgdorferi sensu lato. Infect Immun., 65, 3180-3185. MEDLINE Abstract

Kapitonov ,D. and Yu,R.K. (1997a) Cloning, characterization and expression of human ceramide galactosyltransferase cDNA. Biochem. Biophys. Res. Commun., 232, 449-453. MEDLINE Abstract

Kapitonov ,D. and Yu,R.K. (1997b) Molecular cloning and expression of ceramide galactosyltransferases. Comparison with other glycosyltransferases. In Biochemistry. Virginia Commonwealth University, Richmond.

Kapitonov ,D. and Yu,R.K. (1997c) Molecular cloning and expression of ceramide galactosyltransferases. Comparison with other glycosyltransferases. In Biochemistry and Molecular Biophysics. Medical College of Virginia of Virginia Commonwealth University, Richmond, pp. 179.

Katsutoshi ,S. (1996) Molecular cloning and characterisation of sialyltransferases. Trends Glycosci. Glycotech., 8, 195-215.

Kelly ,R.F. and Whitfield,C. (1996) Clonally diverse rfb gene clusters are involved in expression of a family of related d-galactan O antigens in Klebsiella species. J. Bacteriol., 178, 5205-5214. MEDLINE Abstract

Khan ,A.S., Johnston,N.C., Goldfine,H. and Schifferli,D.M. (1996) Porcine 987P glycolipid receptors on intestinal brush borders and their cognate bacterial ligands. Infect. Immun., 64, 3688-3693. MEDLINE Abstract

Kido ,N., Torgov,V.I., Sugiyama,T., Uchiya,K., Sugihara,H., Komatsu,T., Kato,N. and Jann,K. (1995) Expression of the O9 polysaccharide of Escherichia coli: sequencing of the E.coli O9 rfb gene cluster, characterization of mannosyl transferases and evidence for an ATP-binding cassette transport system. J. Bacteriol., 177, 2178-2187. MEDLINE Abstract

Kimura ,T. and Owens,I.S. (1987) Mouse UDP glucuronosyltransferase. cDNA and complete amino acid sequence and regulation. Eur. J. Biochem., 168, 515-521. MEDLINE Abstract

Korolik ,V., Fry,B.N., Alderton,M.R., van der Zeijst,B.A. and Coloe,P.J. (1997) Expression of Campylobacter hyoilei lipo-oligosaccharide (LOS) antigens in Escherichia coli. Microbiology, 143, 3481-3489. MEDLINE Abstract

Kutish ,G.F., Li,Y., Lu,Z., Furuta,M., Rock,D.L. and Van Etten,J.L. (1996) Analysis of 76 kb of the chlorella virus PBCV-1 330-kb genome: map positions 182 to 258. Virology, 223, 303-317. MEDLINE Abstract

Lannert ,H., Bunning,C., Jeckel,D. and Wieland,F.T. (1994) Lactosylceramide is synthesized in the lumen of the Golgi apparatus. FEBS Lett., 342, 91-96. MEDLINE Abstract

Lazard ,D., Tal,N., Rubinstein,M., Khen,M., Lancet,D. and Zupko,K. (1990) Identification and biochemical analysis of novel olfactory-specific cytochrome P-450IIA and UDP-glucuronosyl transferase. Biochemistry, 29, 7433-7440. MEDLINE Abstract

Lazarevic ,V., Mauel,C., Soldo,B., Freymond,P.P., Margot,P. and Karamata,D. (1995) Sequence analysis of the 308 degrees to 311 degrees segment of the Bacillus subtilis 168 chromosome, a region devoted to cell wall metabolism, containing non-coding grey holes which reveal chromosomal rearrangements. Microbiology, 141, 329-335. MEDLINE Abstract

Lee ,C.Y. (1995) Association of staphylococcal type-1 capsule-encoding genes with a discrete genetic element. Gene, 167, 115-119. MEDLINE Abstract

Lee ,Y.C., Kojima,N., Wada,E., Kurosawa,N., Nakaoka,T., Hamamoto,T. and Tsuji,S. (1994) Cloning and expression of cDNA for a new type of Gal[beta]1,3GalNAc [alpha]2,3-sialyltransferase. J. Biol. Chem., 269, 10028-10033. MEDLINE Abstract

Lerouge ,P., Roche,P., Faucher,C., Maillet,F., Truchet,G., Prome,J.C. and Denarie,J. (1990) Symbiotic host-specificity of Rhizobium meliloti is determined by a sulphated and acylated glucosamine oligosaccharide signal. Nature, 344, 781-784. MEDLINE Abstract

Lin ,W.S., Cunneen,T. and Lee,C.Y. (1994) Sequence analysis and molecular characterization of genes required for the biosynthesis of type 1 capsular polysaccharide in Staphylococcus aureus. J. Bacteriol., 176, 7005-7016. MEDLINE Abstract

Liu ,D., Haase,A.M., Lindqvist,L., Lindberg,A.A. and Reeves,P.R. (1993) Glycosyl transferases of O-antigen biosynthesis in Salmonella enterica: identification and characterization of transferase genes of groups B, C2 and E1. J. Bacteriol., 175, 3408-3413. MEDLINE Abstract

Mackenzie ,P.I. (1986) Rat liver UDP-glucuronosyltransferase. cDNA sequence and expression of a form glucuronidating 3-hydroxyandrogens. J. Biol. Chem., 261, 14112-14117. MEDLINE Abstract

Mackenzie ,P.I. (1987) Rat liver UDP-glucuronosyltransferase. Identification of cDNAs encoding two enzymes which glucuronidate testosterone, dihydrotestosterone and beta-estradiol. J. Biol. Chem., 262, 9744-9749. MEDLINE Abstract

Mackenzie ,P.I. (1990) The cDNA sequence and expression of a variant 17 beta-hydroxysteroid UDP-glucuronosyltransferase. J. Biol. Chem., 265, 8699-8703. MEDLINE Abstract

Mackenzie ,P.I., Owens,I.S., Burchell,B., Bock,K.W., Bairoch,A., Belanger,A., Fournel-Gigleux,S., Green,M., Hum,D.W., Iyanagi,T., Lancet,D., Louisot,P., Magdalou,J., Chowdhury,J.R., Ritter,J.K., Schachter,H., Tephly,T.R., Tipton,K.F. and Nebert,D.W. (1997) The UDP glycosyltransferase gene superfamily: recommended nomenclature update based on evolutionary divergence. Pharmacogenetics, 7, 255-269. MEDLINE Abstract

Madduri ,K. and Hutchinson,C.R. (1995) Functional characterization and transcriptional analysis of a gene cluster governing early and late steps in daunorubicin biosynthesis in Streptomyces peucetius. J. Bacteriol., 177, 3879-3884. MEDLINE Abstract

Mauel ,C., Young,M., Monsutti-Grecescu,A., Marriott,S.A. and Karamata,D. (1994) Analysis of Bacillus subtilis tag gene expression using transcriptional fusions. Microbiology, 140, 2279-2288. MEDLINE Abstract

Meech ,R. and Mackenzie,P.I. (1997) Structure and function of uridine diphosphate glucuronosyltransferases. Clin. Exp. Pharmacol. Physiol., 24, 907-915. MEDLINE Abstract

Mengin-Lecreulx ,D., Texier,L., Rousseau,M. and van Heijenoort,J. (1991) The murG gene of Escherichia coli codes for the UDP-N-acetylglucosamine: N-acetylmuramyl- (pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase involved in the membrane steps of peptidoglycan synthesis. J. Bacteriol., 173, 4625-4636. MEDLINE Abstract

Mirsky ,R., Winter,J., Abney,E.R., Pruss,R.M., Gavrilovic,J. and Raff,M.C. (1980) Myelin-specific proteins and glycolipids in rat Schwann cells and oligodendrocytes in culture. J. Cell Biol., 84, 483-494. MEDLINE Abstract

Mitchison ,M., Bulach,D.M., Vinh,T., Rajakumar,K., Faine,S. and Adler,B. (1997) Identification and characterization of the dTDP-rhamnose biosynthesis and transfer genes of the lipopolysaccharide-related rfb locus in Leptospira interrogans serovar Copenhageni. J. Bacteriol., 179, 1262-1267. MEDLINE Abstract

Morona ,R., Macpherson,D.F., Van Den Bosch,L., Carlin,N.I. and Manning,P.A. (1995) Lipopolysaccharide with an altered O-antigen produced in Escherichia coli K-12 harbouring mutated, cloned Shigella flexneri rfb genes. Mol. Microbiol., 18, 209-223. MEDLINE Abstract

Norqvist ,A. and Wolf-Watz,H. (1993) Characterization of a novel chromosomal virulence locus involved in expression of a major surface flagellar sheath antigen of the fish pathogen Vibrio anguillarum. Infect. Immun., 61, 2434-2444. MEDLINE Abstract

Ochsner ,U.A., Fiechter,A. and Reiser,J. (1994) Isolation, characterization and expression in Escherichia coli of the Pseudomonas aeruginosa rhlAB genes encoding a rhamnosyltransferase involved in rhamnolipid biosurfactant synthesis. J. Biol. Chem., 269, 19787-19795. MEDLINE Abstract

O'Reilly ,D., Brown,M. and Miller,L. (1992) Alteration of ecdysteroid metabolism due to baculovirus infection of the fall armyworm Spodoptera frudiperda: host ecdysteroids are conjugated with galactose. Insect Biochem. Mol. Biol., 22, 313-320.

O'Reilly ,D.R. and Miller,L.K. (1989) A baculovirus blocks insect molting by producing ecdysteroid UDP-glucosyl transferase. Science, 245, 1110-1112. MEDLINE Abstract

Orlean ,P. (1990) Dolichol phosphate mannose synthase is required in vivo for glycosyl phosphatidylinositol membrane anchoring, O mannosylation and N glycosylation of protein in Saccharomyces cerevisiae. Mol. Cell Biol., 10, 5796-5805. MEDLINE Abstract

Orlean ,P., Albright,C. and Robbins,P.W. (1988) Cloning and sequencing of the yeast gene for dolichol phosphate mannose synthase, an essential protein. J. Biol. Chem., 263, 17499-17507. MEDLINE Abstract

Otten ,S.L., Liu,X., Ferguson,J. and Hutchinson,C.R. (1995) Cloning and characterization of the Streptomyces peucetius dnrQS genes encoding a daunosamine biosynthesis enzyme and a glycosyl transferase involved in daunorubicin biosynthesis. J. Bacteriol., 177, 6688-6692. MEDLINE Abstract

Paulson ,J.C. and Colley,K.J. (1989) Glycosyltransferases. Structure, localization and control of cell type- specific glycosylation. J. Biol. Chem., 264, 17615-17618. MEDLINE Abstract

Pearson ,M.N., Bjornson,R.M., Ahrens,C. and Rohrmann,G.F. (1993) Identification and characterization of a putative origin of DNA replication in the genome of a baculovirus pathogenic for Orgyia pseudotsugata. Virology, 197, 715-725. MEDLINE Abstract

Petit ,C., Rigg,G.P., Pazzani,C., Smith,A., Sieberth,V., Stevens,M., Boulnois,G., Jann,K. and Roberts,I.S. (1995) Region 2 of the Escherichia coli K5 capsule gene cluster encoding proteins for the biosynthesis of the K5 polysaccharide. Mol. Microbiol., 17, 611-620. MEDLINE Abstract

Pradel ,E., Parker,C.T. and Schnaitman,C.A. (1992) Structures of the rfaB, rfaI, rfaJ and rfaS genes of Escherichia coli K-12 and their roles in assembly of the lipopolysaccharide core. J. Bacteriol., 174, 4736-4745. MEDLINE Abstract

Price ,N.P., Relic,B., Talmont,F., Lewin,A., Prome,D., Pueppke,S.G., Maillet,F., Denarie,J., Prome,J.C. and Broughton,W.J. (1992) Broad-host-range Rhizobium species strain NGR234 secretes a family of carbamoylated and fucosylated, nodulation signals that are O-acetylated or sulphated. Mol. Microbiol, 6, 3575-3584. MEDLINE Abstract

Rahman ,M.M., Guard-Petter,J., Asokan,K. and Carlson,R.W. (1997) The structure of the capsular polysaccharide from a swarming strain of pathogenic Proteus vulgaris. Carbohydr. Res., 301, 213-220. MEDLINE Abstract

Reeves ,P. (1993) Evolution of Salmonella O antigen variation by interspecific gene transfer on a large scale. Trends Genet., 9, 17-22. MEDLINE Abstract

Reinhold ,B.B., Chan,S.Y., Reuber,T.L., Marra,A., Walker,G.C. and Reinhold,V.N. (1994) Detailed structural characterization of succinoglycan, the major exopolysaccharide of Rhizobium meliloti Rm1021. J. Bacteriol., 176, 1997-2002. MEDLINE Abstract

Relic ,B., Perret,X., Estrada-Garcia,M.T., Kopcinska,J., Golinowski,W., Krishnan,H.B., Pueppke,S.G. and Broughton,W.J. (1994) Nod factors of Rhizobium are a key to the legume door. Mol. Microbiol., 13, 171-178. MEDLINE Abstract

Reuber ,T.L. and Walker,G.C. (1993) Biosynthesis of succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti. Cell, 74, 269-280. MEDLINE Abstract

Riegel ,C.I., Lanner-Herrera,C. and Slavicek,J.M. (1994) Identification and characterization of the ecdysteroid UDP-glucosyltransferase gene of the Lymantria dispar multinucleocapsid nuclear polyhedrosis virus. J. Gen. Virol., 75, 829-838. MEDLINE Abstract

Ritter ,J.K., Sheen,Y.Y. and Owens,I.S. (1990) Cloning and expression of human liver UDP-glucuronosyltransferase in COS-1 cells. 3,4-Catechol estrogens and estriol as primary substrates. J. Biol. Chem., 265, 7900-7906. MEDLINE Abstract

Ritter ,J.K., Chen,F., Sheen,Y.Y., Lubet,R.A. and Owens,I.S. (1992a) Two human liver cDNAs encode UDP-glucuronosyltransferases with 2 log differences in activity toward parallel substrates including hyodeoxycholic acid and certain estrogen derivatives. Biochemistry, 31, 3409-3414. MEDLINE Abstract

Ritter ,J.K., Chen,F., Sheen,Y.Y., Tran,H.M., Kimura,S., Yeatman,M.T. and Owens,I.S. (1992b) A novel complex locus UGT1 encodes human bilirubin, phenol and other UDP-glucuronosyltransferase isozymes with identical carboxyl termini. J. Biol. Chem., 267, 3257-3261. MEDLINE Abstract

Sato ,H., Aono,S., Kashiwamata,S. and Koiwai,O. (1991) Genetic defect of bilirubin UDP-glucuronosyltransferase in the hyperbilirubinemic Gunn rat. Biochem. Biophys. Res. Commun., 177, 1161-1164. MEDLINE Abstract

Sau ,S. and Lee,C.Y. (1996) Cloning of type 8 capsule genes and analysis of gene clusters for the production of different capsular polysaccharides in Staphylococcus aureus. J. Bacteriol., 178, 2118-2126. MEDLINE Abstract

Schaeren-Wiemers ,N., van der Bijl,P. and Schwab,M.E. (1995) The UDP-galactose:ceramide galactosyltransferase: expression pattern in oligodendrocytes and Schwann cells during myelination and substrate preference for hydroxyceramide. J. Neurochem, 65, 2267-2278. MEDLINE Abstract

Schulte ,S. and Stoffel,W. (1993) Ceramide UDPgalactosyltransferase from myelinating rat brain: purification, cloning and expression. Proc. Natl. Acad. Sci. USA, 90, 10265-10269. MEDLINE Abstract

Schultze ,M., Quiclet-Sire,B., Kondorosi,E., Virelizer,H., Glushka,J.N., Endre,G., Gero,S.D. and Kondorosi,A. (1992) Rhizobium meliloti produces a family of sulfated lipooligosaccharides exhibiting different degrees of plant host specificity. Proc. Natl. Acad. Sci. USA, 89, 192-196. MEDLINE Abstract

Schutzbach ,J.S. and Zimmerman,J.W. (1992) Yeast dolichyl-phosphomannose synthase: reconstitution of enzyme activity with phospholipids. Biochem. Cell Biol., 70, 460-465. MEDLINE Abstract

Schutzbach ,J.S., Zimmerman,J.W. and Forsee,W.T. (1993) The purification and characterization of recombinant yeast dolichyl-phosphate-mannose synthase. Site-directed mutagenesis of the putative dolichol recognition sequence. J. Biol. Chem., 268, 24190-24196. MEDLINE Abstract

Scotti ,C. and Hutchinson,C.R. (1996) Enhanced antibiotic production by manipulation of the Streptomyces peucetius dnrH and dnmT genes involved in doxorubicin (adriamycin) biosynthesis. J. Bacteriol., 178, 7316-7321. MEDLINE Abstract

Shimojima ,M., Ohta,H., Iwamatsu,A., Masuda,T., Shioi,Y. and Takamiya,K. (1997) Cloning of the gene for monogalactosyldiacylglycerol synthase and its evolutionary origin. Proc. Natl. Acad. Sci. USA, 94, 333-337. MEDLINE Abstract

Silakowski ,B., Pospiech,A., Neumann,B. and Schairer,H.U. (1996) Stigmatella aurantiaca fruiting body formation is dependent on the fbfA gene encoding a polypeptide homologous to chitin synthases. J. Bacteriol., 178, 6706-6713. MEDLINE Abstract

Skurnik ,M., Venho,R., Toivanen,P. and al-Hendy,A. (1995) A novel locus of Yersinia enterocolitica serotype O:3 involved in lipopolysaccharide outer core biosynthesis. Mol. Microbiol, 17, 575-594. MEDLINE Abstract

Solenberg ,P.J., Matsushima,P., Stack,D.R., Wilkie,S.C., Thompson,R.C. and Baltz,R.H. (1997) Production of hybrid glycopeptide antibiotics in vitro and in Streptomyces toyocaensis. Chem. Biol., 4, 195-202. MEDLINE Abstract

Stahl ,N., Jurevics,H., Morell,P., Suzuki,K. and Popko,B. (1994) Isolation, characterization and expression of cDNA clones that encode rat UDP-galactose: ceramide galactosyltransferase. J. Neurosci. Res., 38, 234-242. MEDLINE Abstract

Standal ,R., Iversen,T.G., Coucheron,D.H., Fjaervik,E., Blatny,J.M. and Valla,S. (1994) A new gene required for cellulose production and a gene encoding cellulolytic activity in Acetobacter xylinum are colocalized with the bcs operon [published erratum appears in J. Bacteriol., 1994 176 (11), 3443]. J. Bacteriol., 176, 665-672. MEDLINE Abstract

Stingele ,F., Neeser,J.R. and Mollet,B. (1996) Identification and characterization of the eps (Exopolysaccharide) gene cluster from Streptococcus thermophilus Sfi6. J. Bacteriol., 178, 1680-1690. MEDLINE Abstract

Sulston ,J., Du,Z., Thomas,K., Wilson,R., Hillier,L., Staden,R., Halloran,N., Green,P., Thierry-Mieg,J., Qiu,L., et al. (1992) The C.elegans genome sequencing project: a beginning [see comments]. Nature, 356, 37-41. MEDLINE Abstract

Toniolo ,A., Serra,C., Conaldi,P.G., Basolo,F., Falcone,V. and Dolei,A. (1995) Productive HIV-1 infection of normal human mammary epithelial cells. AIDS, 9, 859-866. MEDLINE Abstract

Tukey ,R.H., Pendurthi,U.R., Nguyen,N.T., Green,M.D. and Tephly,T.R. (1993) Cloning and characterization of rabbit liver UDP-glucuronosyltransferase cDNAs. Developmental and inducible expression of 4-hydroxybiphenyl UGT2B13. J. Biol. Chem., 268, 15260-15266. MEDLINE Abstract

Ueda ,T., Suga,Y., Yahiro,N. and Matsuguchi,T. (1995) Phylogeny of Sym plasmids of rhizobia by PCR-based sequencing of a nodC segment. J. Bacteriol., 177, 468-472. MEDLINE Abstract

van der Bijl ,P., Strous,G.J., Lopes-Cardozo,M., Thomas-Oates,J. and van Meer,G. (1996) Synthesis of non-hydroxy-galactosylceramides and galactosyldiglycerides by hydroxy-ceramide galactosyltransferase. Biochem. J., 317, 589-597. MEDLINE Abstract

van Kranenburg ,R., Marugg,J.D., van,S.,II, Willem,N.J. and de Vos,W.M. (1997) Molecular characterization of the plasmid-encoded eps gene cluster essential for exopolysaccharide biosynthesis in Lactococcus lactis. Mol. Microbiol., 24, 387-397. MEDLINE Abstract

Watson ,A., Smaldon,N., Lucke,R. and Hawkins,T. (1993) The Caenorhabditis elegans genome sequencing project: first steps in automation. Nature, 362, 569-570. MEDLINE Abstract

Wilson ,R., Ainscough,R., Anderson,K., Baynes,C., Berks,M., Bonfield,J., Burton,J., Connell,M., Copsey,T., Cooper,J., et al. (1994) 2.2 Mb of contiguous nucleotide sequence from chromosome III of C.elegans [see comments]. Nature, 368, 32-38. MEDLINE Abstract

Wong ,H.C., Fear,A.L., Calhoon,R.D., Eichinger,G.H., Mayer,R., Amikam,D., Benziman,M., Gelfand,D.H., Meade,J.H., Emerick,A.W. and et al. (1990) Genetic organization of the cellulose synthase operon in Acetobacter xylinum. Proc. Natl. Acad. Sci. USA, 87, 8130-8134. MEDLINE Abstract

Xiang ,S.H., Haase,A.M. and Reeves,P.R. (1993) Variation of the rfb gene clusters in Salmonella enterica. J. Bacteriol., 175, 4877-4884. MEDLINE Abstract

Xiang ,S.H., Hobbs,M. and Reeves,P.R. (1994) Molecular analysis of the rfb gene cluster of a group D2 Salmonella enterica strain: evidence for its origin from an insertion sequence-mediated recombination event between group E and D1 strains. J. Bacteriol., 176, 4357-4365. MEDLINE Abstract

Yahi ,N., Baghdiguian,S., Moreau,H. and Fantini,J. (1992) Galactosyl ceramide (or a closely related molecule) is the receptor for human immunodeficiency virus type 1 on human colon epithelial HT29 cells. J. Virol, 66, 4848-4854. MEDLINE Abstract

Yahi ,N., Sabatier,J.M., Baghdiguian,S., Gonzalez-Scarano,F. and Fantini,J. (1995) Synthetic multimeric peptides derived from the principal neutralization domain (V3 loop) of human immunodeficiency virus type 1 (HIV-1) gp120 bind to galactosylceramide and block HIV-1 infection in a human CD4- negative mucosal epithelial cell line. J. Virol, 69, 320-325. MEDLINE Abstract

Ye ,J., Dickens,M.L., Plater,R., Li,Y., Lawrence,J. and Strohl,W.R. (1994) Isolation and sequence analysis of polyketide synthase genes from the daunomycin-producing Streptomyces sp. strain C5. J. Bacteriol., 176, 6270-6280. MEDLINE Abstract

Zhang ,L., al-Hendy,A., Toivanen,P. and Skurnik,M. (1993) Genetic organization and sequence of the rfb gene cluster of Yersinia enterocolitica serotype O:3: similarities to the dTDP-l-rhamnose biosynthesis pathway of Salmonella and to the bacterial polysaccharide transport systems. Mol. Microbiol., 9, 309-321. MEDLINE Abstract


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