©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of Chitin Synthase 2 of Saccharomyces cerevisiae
IMPLICATION OF TWO HIGHLY CONSERVED DOMAINS AS POSSIBLE CATALYTIC SITES (*)

Shigehisa Nagahashi (1), Masayuki Sudoh (1), Naomi Ono (1), Rumi Sawada (1), Emi Yamaguchi (1), Yukiko Uchida (1), Toshiyuki Mio (1), Masamichi Takagi (2), Mikio Arisawa (1), Hisafumi Yamada-Okabe (1)(§)

From the (1) Department of Mycology, Nippon Roche Research Center, 200 Kajiwara, Kamakura, Kanagawa 247 and the (2) Department of Agricultural Chemistry, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Chitin synthase 2 of Saccharomyces cerevisiae was characterized by means of site-directed mutagenesis and subsequent expression of the mutant enzymes in yeast cells. Chitin synthase 2 shares a region whose sequence is highly conserved in all chitin synthases. Substitutions of conserved amino acids in this region with alanine (alanine scanning) identified two domains in which any conserved amino acid could not be replaced by alanine to retain enzyme activity. These two domains contained unique sequences, Glu-Asp-Arg and Gln-Arg-Arg-Arg-Trp, that were conserved in all types of chitin synthases. Glu or arginine at 563, 602, and 603 could be substituted by glutamic acid and lysine, respectively, without significant loss of enzyme activity. However, even conservative substitutions of Asp with glutamic acid, Gln with asparagine, Arg with lysine, or Trp with tyrosine drastically decreased the activity, but did not affect apparent K values for the substrate significantly. In addition to these amino acids, Asp was also found in all chitin synthase. The mutant harboring a glutamic acid substitution for Asp severely lost activity, but it showed a similar apparent K value for the substrate. Amounts of the mutant enzymes in total membranes were more or less the same as found in the wild type. Furthermore, Asp, Asp, Gln, Arg, and Trp are completely conserved in other proteins possessing N-acetylglucosaminyltransferase activity such as NodC proteins of Rhizobium bacterias. These results suggest that Asp, Asp, Gln, Arg, and Trp are located in the active pocket and that they function as the catalytic residues of the enzyme.


INTRODUCTION

Chitin, a -1,4-linked polymer of N-acetylglucosamine, is one of the components of the yeast cell wall and is synthesized by the enzyme called chitin synthase (1) . Three chitin synthase genes have been identified in the yeasts Saccharomyces cerevisiae(2, 3, 4, 5) and Candida albicans(6, 7, 8) , and they are designated as CHS1, CHS2, and CHS3 (CHS3 is called as CAL1/CSD2 in S. cerevisiae) (2, 3, 4, 5, 6, 7, 8, 9) . Chitin synthase 1 (Chs1p) and Chs2p are zymogens (10, 11, 12) , but Chs3p is not simply activated by proteinases (4, 13, 14) ; Chs3p shows zymogenic property only when it is treated with proteinases in the presence of the substrate, UDP-GlcNAc (15) . Several lines of evidence, including the phenotypes of cells in which one of the chitin synthase genes is disrupted, suggest a functional distinction for each chitin synthase in S. cerevisiae; Chs1p repairs damaged chitin during cell separation, Chs2p is required for primary septum formation, and Chs3p is involved in all other chitin syntheses (16, 4, 17) . Although none of chitin synthase genes is so far reported to be essential for the vegetative growth of yeast cells (2, 17, 18) , combinational disruption of CHS2 and CAL1 is lethal in S. cerevisiae(17) , suggesting that Chs1p can not functionally complement Chs2p and Chs3p.

Chs1p, the most abundant enzyme in S. cerevisiae cells among three chitin synthases, is solubilized from a particulate fraction with digitonin and is partially purified through gel filtration followed by product entrapment (11) . Molecular mass of the partially purified Chs1p is about 570 kDa in which a 63-kDa polypeptide is a major component of the enzyme (11) . The enzyme requires UDP-GlcNAc as the substrate (10, 11) , and although GlcNAc is an activator of the enzyme, the real role of GlcNAc remains to be established (10, 11) . Chs2p has been characterized in the membrane fraction of S. cerevisiae cells lacking CHS1 and is shown to be much less abundant than Chs1p (the Chs2p level is estimated to be about 5% of that of Chs1p) (12) . Although Chs1p and Chs2p share sequence similarity and many characteristics such as membrane localization, activation by GlcNAc, use of UDP-GlcNAc as the substrate, and K for the substrate, they show different specificity for divalent cations and different pH optima (10, 12) . CHS1 seems not to be a critical gene for chitin synthesis in yeast cells because S. cerevisiae cells lacking CHS1 grow normally without showing apparent loss of cell wall chitin content in the absence of Chs1p activity (2) . On the other hand, disruption of CHS2 causes severe growth defects and morphological abnormalities (17) , suggesting that Chs2p plays more important roles in chitin synthesis in vivo and in cell growth than Chs1p.

Despite the characterization of chitin synthase activities and cloning of the chitin synthase genes, no information is available about the protein domains essential for enzyme activity such as active sites, probably due to the difficulties in overexpressing and purifying the enzyme in large quantities. Here we report the highly conserved region of Chs2p and the possible involvement of this region in the catalytic reaction by chitin synthase is proposed.


MATERIALS AND METHODS

Yeast Strains and Plasmids

Haploid S. cerevisiae strain, R27-7C-1C (a trp1 leu2 ura3 his3), was used as the wild type strain. RRA400 (a trp1 leu2 ura3 his3 cal1::HIS3) was a derivative of R27-7C-1C in which CAL1 was replaced by HIS3 using YIp plasmid (19) carrying CAL1 and HIS3. In RRA400, CHS1 was further replaced by URA3 using YIp plasmid (19) carrying CHS1 and URA3 to generate RRA400-1U (a trp1 leu2 ura3 his3 chs1::URA3 cal1::HIS3) which harbored neither CHS1 nor CAL1. Cells of the above strains were cultured at 30 °C with yeast nitrogen base supplemented with glucose and required amino acids. Expression of chitin synthase was induced by transferring the cells in early logarithmic phase to the medium containing galactose and further culturing the cells for 12 h at 30 °C.

A 0.8-kb()BamHI-EcoRI fragment containing GAL1 promoter and a 0.2- kb XbaI-HindIII fragment containing GAP terminator were excised from pYPR3831 (20) and ligated at the HindIII cleavage site of YEp351 (21) in which unique SalI site had been already destroyed. The resulting plasmid harboring GAL1 promoter, GAP terminator, 2-µm replication origin, and LEU2 as a selectable marker was designated as YpLX. To overexpress Chs2p in S. cerevisiae cells, CHS2 was introduced downstream of the GAL1 promoter. 2.9-kb DNA fragment containing the entire coding region of S. cerevisiae CHS2 was amplified by polymerase chain reaction. The primers used for the polymerase chain reaction were 5`-GACTCTAGAATGACGAGAAACCCG-3` and 5`-GATGCGGCATCTAGATTAGCCCTTTTTGTGGAA-3` which possessed XbaI cleavage site in addition to the corresponding sequences to CHS2. The resulting DNA was ligated at the XbaI cleavage site of YpLX which was located at the junction of GAL1 promoter and terminator. The resulting plasmid, YpLCS2, was then transfected into the yeast cells by electroporation as described (22) , and leucine prototrophs were collected and used for the experiments.

Site-directed Mutagenesis

A series of CHS2 mutants were generated by site-directed mutagenesis using uracil-containing single-stranded DNA as described by Kunkel et al.(23, 24) . A 1.6-kb PstI/SalI fragment of CHS2 was cloned in pUC118 and used for generating uracil-containing single stranded DNA. DNA fragments of CHS2 harboring the particular mutations were ligated at PstI-SalI cleavage site of YpLCS2 and transfected into S. cerevisiae cells. All mutations were confirmed by sequencing the DNA as described elsewhere (25) .

Assay of Chitin Synthase

S. cerevisiae cells with or without the induction of chitin synthases were harvested and washed twice with 20 mM Tris-Cl, pH 7.5, and suspended in 1 ml/g yeast of a buffer containing 20 mM Tris-Cl, pH 7.5, 0.25 mM phenylmethylsulfonyl fluoride, 2 µg/ml chymostatin, 1.5 µg/ml leupeptin, 1 µg/ml pepstatin, and 5 µg/ml antipain. Cells were lysed with glass beads, and cell debris were removed by the low speed centrifugation. The membrane was then sedimented at 100,000 g for 50 min at 4 °C, washed once with 20 mM Tris-Cl, pH 7.5, suspended in a buffer containing 20 mM Tris-Cl, pH 7.5, and 33% glycerol, and stored at -80 °C until use. Chs2p assay was carried out according to the method of Sburlati and Cabib (12) in a standard 100-µl reaction mixture containing 30 mM Tris-Cl, pH 7.5, 2.5 mM Co(CHCOO), 32 mM GlcNAc, 0.1 mM [H]UDP-GlcNAc (specific activity, 95,880 disintegrations/min/nmol), 10-50 µg of protein of yeast membrane fraction at 30 °C for 60 min.

Generation of Antibody

A polyclonal antibody was raised against a part of Chs2p (from amino acid positions 192 to 624) which was expressed in insect cells (Sf21 cells) as a fusion protein with a 6-histidine tail (26) . In this fusion protein, the 6 histidines were linked to the C-terminal end. A 1.3-kb fragment of CHS2 encoding 433 amino acids (from amino acid positions 192-624 of Chs2p) was amplified by polymerase chain reaction using CHS2 gene as a template, and ligated at the BamHI-XhoI cleavage site of pBacPAK9 (27) . Primers used for amplifying a part of CHS2 gene were 5`-CGGCGGATCCAAATGTCTGCAGACACTTTCAATGAAACA-3` and 5`-CCGGCCTCGAGCCAAATTTGGTAGAAATGCAATTGAGCA-3`. The resulting plasmid harboring a part of CHS2 gene was then digested with XhoI and ligated with an oligonucleotide coding for 6 histidines to generate pB9CS2(192-624)His. pB9CS2(192-624)His was transfected into Sf21 cells together with BacPAK6 viral DNA using lipofectin (27) . Sf21 cells were harvested 72 h post-infection, and the insoluble Chs2p-6-histidine fusion protein was extracted by lysing cells with 6 M guanidine-HCl, purified by Nickel chelating column chromatography through a linear gradient of immidazole (0-500 mM) in 6 M guanidine-HCl, precipitated by trichloroacetic acid, and injected into rabbits subcutaneously. IgG fractions containing anti-Chs2p antibody were obtained from crude sera by ammonium sulfate precipitation followed by Protein A-Superose column chromatography.

Western Blotting

20 µg of protein of total yeast membranes prepared as mentioned above were fractionated on 8% SDS-polyacrylamide gel, transferred to a PVDF membrane electrophoretically, Western blotted with anti-Chs2p polyclonal antibody, and then with horseradish peroxidase-conjugated anti-rabbit IgG. Chs2p was visualized by incubating a PVDF membrane with cyclic diacylhydrazides (ECL detection kit, Amersham) and subsequent exposure to an x-ray film (Kodak). Quantification of Chs2p in the total membrane was carried out by comparing the density of Chs2p in the total membrane with that of the purified Chs2p expressed in Sf21 cells using a densitometer.


RESULTS

When the amino acid sequence of S. cerevisiae Chs2p was compared to those of other chitin synthases, we found that a region comprising amino acid positions 490-607 was highly conserved in all types of chitin synthases of yeasts as well as mycelial fungi (Fig. 1A; see also Refs. 2-8 and 28). This region was, therefore, designated as Con1 (conserved region 1). Con1 could be divided into three subdomains based on the frequencies of the appearance of conserved amino acids (Fig. 1B). The sequence of Chs3p became rather diverse in the N-terminal region outside Con1, whereas Chs1p and Chs2p still shared high sequence similarity. Further N-terminal and C-terminal regions outside Con1 showed much lower sequence similarity even between Chs1p and Chs2p. Since Chs1p, 2p, and 3p perform the same catalytic function, the active site of these enzymes would be expected to be conserved. From this point of view, it is likely that Con1 contains the active site of chitin synthase. In order to address this possibility, we generated a series of mutant enzymes in which one of the conserved amino acids in Con1 was replaced by alanine.


Figure 1: Highly conserved region of Chs2p. A, location of highly conserved region of Chs2p. Con1 represents the highly conserved region of Chs2p with brackets indicating subdomains in this region. Numbers in the upper side of the bar indicate the position of amino acids from the N-terminal end of Chs2p. B, comparison of the amino acid sequence of Con1 with several chitin synthases. Amino acids drawn with bold characters are those conserved in all chitin synthases listed here. I-III represent subdomains where conserved amino acids appear at high frequencies. TM, potential transmembrane domains; ScChs1-3, S. cerevisiae chitin synthase 1-3; CaChs1-3, C, albicans chitin synthase 1-3; RoChs1-3, R. oligosporus chitin synthase 1-3 (40); AnChsA, B, A. nidulans chitin synthase A, B.



Chs2p could not be expressed in bacterial or insect cells in an active form. Therefore, a series of plasmid DNAs carrying mutant CHS2 genes whose expression was under the control of GAL1 promoter was introduced into the yeast strain, RRA400-1U, in which endogenous CHS1 and CAL1 had been disrupted, and expression of the mutant enzymes were induced by culturing the cells in medium containing galactose. When YpLCS2 was introduced into RRA400-1U, Chs2p activity was completely dependent on galactose, and endogenous Chs2p activity was barely detected, indicating that the endogenous Chs2p activity was extremely low and could be neglected in this system (data not shown).

Activities of each mutant enzyme were determined using total membrane fractions. As shown in Fig. 2, substitution of Tyr, Glu, Asp, Arg, Gln, Arg, Arg, Arg, or Trp with alanine resulted in almost complete loss of the activity, whereas alanine substitution of other amino acids retained activities. It was also demonstrated in the control experiments that the amino acids which were not highly conserved (Asn, Ser, Asp, Asp, Asn, Arg, His) could be substituted by alanine without affecting the enzyme activity significantly (Fig. 2).


Figure 2: Effects of amino acid substitutions in Con1 on the activity of Chs2p. Conserved amino acids in Con1 were substituted by alanine, and the effect on enzyme activity is shown in the left panel. The effect of substituting non-conserved amino acids with alanine also are indicated in the right panel. Activities of these mutant enzymes were determined in a standard reaction mixture using 10 µg of the total membrane protein at 30 °C for 60 min.



Domains II and III contained unique sequences, Glu-Asp-Arg and Gln-Arg-Arg-Arg-Trp, respectively. These two sequences are completely conserved in all chitin synthases listed in Fig. 1including newly identified chitin synthases of Rhizopus oligosporus and Aspergillus nidulans. The same sequences were also found in recently reported Neurospora crassa Chs1p and Chs2p (28) , suggesting that they are critical sites for the catalytic activities of chitin synthases. In order to address this possibility, we have substituted the amino acids found in these sequences with the analogous ones. As mentioned above, no amino acid in these sequences could be replaced by alanine to retain activity, but E561D, R563K, R602K, and R603K did retain activity, suggesting that Glu, Arg, Arg, and Arg, while quite important for enzyme activity, may not be in the active site itself. However, even conservative replacements in D562E, Q601N, R604K, and W605Y had very little activity (). With the exception of D562E, which was too low to determine the apparent K value, conservative substitutions for any other amino acid in Glu-Asp-Arg and Gln-Arg-Arg-Arg-Trp did not affect their apparent K values for the substrate significantly (). Even Q601N, R604K, and W605Y exhibited similar apparent K values to that of wild type enzyme despite that enzyme activities of these mutants were about 1% of the wild type enzyme activity.

Next, we examined whether or not the hydroxyl group of Asp was necessary for the catalytic activity of the enzyme. Substitution of asparagine for Asp resulted in the complete loss of the activity (). Interestingly, E561Q also showed very little activity (), indicating that the hydroxyl groups of both Glu and Asp are essential for the enzyme activity. Although individual residues in the stretch of basic arginines in domain III (Arg or Arg) could be replaced by lysine without apparent loss of the enzyme activity, we wondered whether both of these basic amino acids could be replaced by lysine simultaneously. The mutant enzyme harboring lysine substitution of Arg and Arg together lost the enzyme activity. This result implies that Arg and Arg are not located simply to provide basic charges and suggests that they may play some significant roles in catalytic activity.

The above results strongly suggest that Glu-Asp-Arg and Gln-Arg-Arg-Arg-Trp are essential sites for the catalytic activity and that some of the amino acids in these sequences, specifically Asp, Gln, Arg and Trp, may function as the catalytic site of the enzyme. However, we cannot rule out the possibility that a decrease in the activities of the mutant enzymes was the trivial consequence of the lower levels of expression. To address this possibility, a polyclonal antibody was raised against Chs2p (from amino acids 192-624) to examine levels of the expression of all of these mutant enzymes. Western blotting revealed that this antibody recognized a protein with approximately molecular mass of 110 kDa in the membranes prepared from cells overexpressing CHS2 but not in the membranes from the vector-transfected control (Fig. 3). The size of this protein corresponded to that estimated by the deduced amino acid sequence of Chs2p. Furthermore, this antibody precipitated the chitin synthase activity when incubated with partially purified Chs2p by product entrapment (data not shown). All these results indicate that the 110-kDa protein detected by the Western blotting was actually Chs2p. Using a part of Chs2p (from amino acids 192-624) purified from insect cells as a standard, the amount of Chs2p in the total yeast membranes of RRA400-1U harboring YpLCS2 was estimated to be about 60 pg/µg of membrane protein when Chs2p expression was induced by galactose. Western blots of total membranes prepared from cells overexpressing mutant Chs2p revealed no significant difference in amounts of protein between wild type and a series of the mutants except for Y521A (Fig. 4, A and B). The expression level of Y521A was about 20% of that of the wild type enzyme, and this might account for a portion of the reduced activity of Y521A ( Fig. 2and Fig. 4A). These results demonstrate that except for Y521A a decrease in the activity of mutant enzymes was the consequence of decrease in the rate of catalytic reaction, and that no other mutation in Chs2p reduced the level of protein expressed. Considering all the data mentioned above, we conclude that the conserved Glu-Asp-Arg and Gln-Arg-Arg-Arg-Trp regions of Chs2p are essential for catalytic activity and that Asp, Gln, Arg, and Trp are the potential catalytic residues of the enzyme.


Figure 3: Expression of Chs2p in insect cells and generation of antibody against Chs2p. A, the Chs2p protein fragment (from amino acid positions 192-624) was isolated from Sf21 cells as described under ``Materials and Methods.'' The 8% SDS-polyacrylamide gels were stained with Coomassie Brilliant Blue. Lane 1, total extract of uninfected Sf21 cells; lane 2, total extract of infected Sf21 cells; lane 3, purified Chs2p fragment. B, 20 µg of membrane protein prepared from cells of RRA400-1U which were transfected with YpLX (vector) or with YpLCS2 (plasmid carrying CHS2) were fractionated on 8% SDS-polyacrylamide gels, transferred to a PVDF membrane, and visualized by Western blotting with anti-Chs2p antibody. Lane 1, purified Chs2p fragment; lane 2, total membrane of RRA400-1U harboring YpLX; lane 3, total membrane of RRA400-1U harboring YpLCS2.




Figure 4: Comparison of levels of protein in wild type and mutant Chs2p constructs. 20 µg of membrane protein from cells overexpressing wild type as well as mutant Chs2p were subjected to 8% SDS-polyacrylamide gel electrophoresis. After blotting on to a PVDF membrane, Chs2p was visualized by Western blotting. Only the Chs2p bands were indicated. Additional details are under ``Materials and Methods.'' A, levels of mutant Chs2p in which one of the conserved amino acids in Con1 was substituted by alanine. B, levels of mutant Chs2p in which one or two of the amino acids in Glu-Asp-Arg and Gln-Arg-Arg-Arg-Trp were conservatively substituted. C, levels of mutant Chs2p in which Asp was substituted with the indicated amino acid.



GlcNAc is known to be an activator of chitin synthases; millimolar addition of GlcNAc to the reaction mixture increases the enzyme activity by severalfold (9, 11, 12) . We have tested the effect of GlcNAc on some of the mutant enzymes. Enhancement of the activity by GlcNAc was quite high in R563K and R602K (Fig. 5B), whereas those of E561D, Q601N, R603K, R604K, and W605Y were not significantly different from wild type enzyme (Fig. 5A). Although the rate of the activation by GlcNAc was much higher in R602K than in R563K, 240 mM GlcNAc increased the enzyme activity of R563K and R602K by more than 20-fold. At this concentration of GlcNAc (240 mM), only about 5-fold increase in the activity was observed with wild type enzyme (Fig. 5B). The finding that mutations in the two separate regions made the enzymes sensitive to enhanced activation by the same small molecule, GlcNAc, implies that these two region Glu-Asp-Arg and Gln-Arg-Arg-Arg-Trp may be located very close to each other three dimensionally.


Figure 5: Effects of GlcNAc on the activity of Chs2p. Total membranes were prepared from cells of RRA400-1U transfected with YpLCS2 that harbored indicated mutations of CHS2 gene, and Chs2p activity was determined using 10 µg of membrane protein in a standard reaction mixture containing indicated concentrations of GlcNAc.



Recently, Szaniszlo and Momany (29) reported that chitin synthases share sequence homology with a short stretch of amino acids constituting the nucleotide-binding fold of a bacterial periplasmic permease (AraG) and of an adenylate kinase (Adk). In this small region, one aspartic acid is completely conserved in all chitin synthases (Asp in Chs2), AraG and Adk (Fig. 6A; see also Ref. 29). These facts also prompted us to examine the possibility that Asp of Chs2p is a part of the active site. Substitution of Asp of Chs2p with alanine or glutamic acid resulted in nearly a complete loss of the enzyme activity. Although the apparent K value of D441A could not be determined because of its extremely low activity, that of D441E was not significantly different from that of wild type enzyme, and the amount of protein expressed of the above two mutant enzymes was more or less the same as the control Chs2p (Fig. 4C and ). All these results indicate that Asp is also an essential amino acid for the catalytic reaction of the enzyme.


Figure 6: Sequence similarity of chitin synthases and NodC proteins. A, sequences of chitin synthases in the region which is homologous to the nucleotide binding fold of a bacterial periplasmic permease (AraG) and an adenylate kinase (Adk) were compared with those of NodC proteins, cellulose synthase, and DG42 protein. The amino acid drawn in bold corresponds to Asp of S. cerevisiae Chs2p. B, sequences of chitin synthases in domains II and III within Con1 were compared to those of NodC proteins, cellulose synthase, and DG42 protein. Amino acids in bold letters are those that correspond to Glu, Gln, Arg, and Trp of S. cerevisiae Chs2p. SacChs2, S. cerevisiae Chs2p (3); CanChs1, C. albicans Chs1p (5, 6); SacChs1, S. cerevisiae Chs1p (2); SacChs3, S. cerevisiae Cal1p (4); RlvNodC, Rhizobium leguminosarum bv. viciae NodC (41); RfNodC, Rhizobium fredii NodC (42); RmNodC, Rhizobium melioti NodC (33); AcNodC, Azorhizobium caulinodans NodC (43); SpHasA, Streptococcus pyogenes hyaluronan synthase (35, 36); AxCs, Acetobacter xylinum cellulose synthase (37); XlDG42, X. laevis DG42 protein (38).




DISCUSSION

We demonstrated here that Asp, Glu-Asp-Arg and Gln-Arg-Arg-Arg-Trp of Chs2p play essential roles in the catalytic activity of the enzyme, and that among them, Asp, Asp, Gln, Arg, and Trp are potential catalytic residues. Catalytic mechanisms deduced for several enzymes, including glycosidases and glycosyltransferases, suggest that acidic or basic catalysts are involved in the protonation of the substrate (30) . In sucrose:1,3--D-glucan 3--D-glycosyltransferases (GTase I) of Streptococcus sobrinus, the aspartic acid near the N-terminal end (Asp) is identified as a catalytic site and is thought to function as an acidic catalyst (31) . Since Asp and Asp of Chs2p are conserved in all chitin synthases and when substituted by asparagine do not retain enzyme activity, it is very likely that one of these residues (Asp or Asp) is functioning as an acidic catalyst. However, we cannot rule out the possibility that Asp is involved in the substrate recognition and binding because activities of D562A, D562E, and D562N were too low to determine their apparent K values for the substrate. Interestingly, E561Q also showed very little activity (), suggesting that the hydroxyl group of Glu may be required for the efficient ionization of the hydroxyl group of Asp, if Asp indeed is functioning as an acidic catalyst. Arg and Arg are also required for activity; simultaneous substitution of these two arginines with lysines resulted in the complete loss of the activity. Because Arg cannot be substituted even with lysine, it is speculated that Arg may be functioning as a basic catalyst and that two arginines in front of Arg may be required for the efficient ionization of the guanidino group of Arg.

If Asp, Asp, Gln, Arg, and Trp of Chs2p are catalytic residues, they might be also conserved in proteins possessing the same catalytic activity as chitin synthases. NodC proteins of Rhizobium as well as Azorhizobium bacterias share significant sequence homology to chitin synthases (Fig. 6). Further, catalytic activity and the substrate of NodC protein are the same as those of chitin synthases (32) . When sequences of NodC proteins were compared to those of chitin synthases, all NodC proteins were found to have sequences, Glu-Asp-Arg and Gln-Gln-Leu-Arg-Trp, which were identical or quite similar to unique sequences identified in chitin synthases (Glu-Asp-Arg and Gln-Arg-Arg-Arg-Trp) (Fig. 6B; see also Refs. 33 and 34). In these sequences, amino acids essential for the catalytic activity of Chs2p could not be substituted even conservatively, Asp in Glu-Asp-Arg, Gln, the third Arg and Trp in Gln-Arg-Arg-Arg-Trp, were completely conserved in NodC proteins as well. Further, Asp of Chs2p is also found in NodC proteins (Fig. 6A, and see also Ref. 29), although the neighboring sequences of this aspartic acid are not highly conserved between chitin synthases and NodC proteins. All these facts strongly support the idea that Asp, Glu-Asp-Arg, and Gln-Arg-Arg-Arg-Trp of Chs2p are crucial sites for catalytic activity and that among them Asp, Asp, Gln, Arg, and Trp are the potential catalytic residues of the enzyme.

Interestingly, similar sequences to Glu-Asp-Arg and Gln-Arg-Arg-Arg-Trp of Chs2p were also found in hyaluronan synthase of Streptococcus pyogenes(35, 36) , cellulose synthase of Acetobacter xylinum(37) and DG42 of Xenopus laevis(38) . These are Asp-Asp-Arg and Gln-Gln-Asn-Arg-Trp in hyaluronan synthase, Tyr-Asp-Ala and Gln-Arg-Val-Arg-Trp in cellulose synthase, and Asp-Asp-Arg and Gln-Gln-Thr-Arg-Trp in DG42, respectively (Fig. 6). As seen in NodC proteins, Asp in Glu-Asp-Arg, Gln, the third Arg and Trp in Gln-Arg-Arg-Arg-Trp of Chs2p were also conserved in corresponding regions of hyaluronan synthase, cellulose synthase, and DG42 (Fig. 6, and see also Ref. 39). Although catalytic activity of DG42 remains to be established, hyaluronan synthase and cellulose synthase catalyze the polymerization of sugars with -1,4-linkage using UDP-sugar as substrate. Thus, amino acids of the potential catalytic sites of Chs2p may be generally conserved in glycosyltransferases which catalyze the synthesis of oligosaccharides with -1,4-linkages.

  
Table: Characteristics of mutant Chs2



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

The abbreviations used are: kb, kilobase(s); PVDF, polyvinylidene difluoride.


ACKNOWLEDGEMENTS

We thank Dr. O. Shimmi for valuable suggestions and repeated discussions.


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