From the Department of Biochemistry, Umeå
University, S-901 87 Umeå, Sweden and the ¶ Department of
Biochemistry and Biophysics, Stockholm University,
S-106 91 Stockholm, Sweden
Received for publication, March 22, 2001, and in revised form, April 6, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Synthesis of the nonbilayer-prone
Lipids are the local environment for most integral and peripheral
membrane proteins, which often depend on the lipids for optimal
function. The large diversity of lipids and the differences in
composition and properties between membranes have made it difficult to
find out common features of bilayer organization and how lipids and
proteins are cooperating in local processes. Lipid-synthesizing pathways have been mapped for the most common types of lipids, and
several of the corresponding enzymes catalyzing these reactions have
been characterized. However, when it comes to the connection between
regulation of bilayer properties and enzyme structure, very little is
known (1). So far, only a few lipid-synthesizing enzymes have been
crystallized. Which structural properties are involved in the catalytic
mechanism of these lipid enzymes, and how are the membrane properties
sensed (1)?
In the well characterized plasma membrane of Acholeplasma
laidlawii, the lipid composition is regulated in a manner to
maintain (i) lipid phase equilibria, close to a potential
bilayer to nonbilayer transition, (ii) a nearly constant radius of
spontaneous curvature, and (iii) a certain anionic surface charge
density of the lipid bilayer. The synthesis of the major
nonbilayer-prone lipid in this membrane, monoglucosyldiacylglycerol
(MGlcDAG)1 (Scheme
1, step I), plays an important role to
fulfill the two first points above but also the third, since it is
strongly regulated by negatively charged lipids (e.g. the
major in vivo lipid phosphatidylglycerol (PG)) (2). MGlcDAG
is consecutively processed into diglucosyl diacylglycerol (DGlcDAG)
(Scheme 1, step II). Consequently, the formation of this glucolipid, a
transfer of Glc from the donor UDP-Glc to the acceptor lipid
diacylglycerol (DAG) catalyzed by a glucosyltransferase (EC 2.4.1.157)
(3), plays a central part in understanding the total regulation of
lipid syntheses in A. laidlawii membranes. Furthermore,
glycolipids including nonbilayer-prone ones are major constituents in
many cell surface membranes, certain bacterial groups, and most
photosynthetic organelles. Fairly little is known about the synthesis
and regulation of these. Usually, they are made in a separate pathway
(as in A. laidlawii), branching from the conserved one to
anionic phospholipids.
-monoglucosyldiacylglycerol (MGlcDAG) is crucial for bilayer packing
properties and the lipid surface charge density in the membrane of
Acholeplasma laidlawii. The gene for the responsible,
membrane-bound glucosyltransferase (alMGS) (EC 2.4.1.157) was sequenced
and functionally cloned in Escherichia coli, yielding
MGlcDAG in the recombinants. Similar amino acid sequences were encoded
in the genomes of several Gram-positive bacteria (especially
pathogens), thermophiles, archaea, and a few eukaryotes. All of these
contained the typical EX7E catalytic motif of the CAZy
family 4 of
-glycosyltransferases. The synthesis of MGlcDAG by a
close sequence analog from Streptococcus pneumoniae (spMGS)
was verified by polymerase chain reaction cloning, corroborating a
connection between sequence and functional similarity for these proteins. However, alMGS and spMGS varied in dependence on anionic phospholipid activators phosphatidylglycerol and cardiolipin, suggesting certain regulatory differences. Fold predictions strongly indicated a similarity for alMGS (and spMGS) with the two-domain structure of the E. coli MurG cell envelope
glycosyltransferase and several amphipathic membrane-binding segments
in various proteins. On the basis of this structure, the alMGS sequence
charge distribution, and anionic phospholipid dependence, a model for
the bilayer surface binding and activity is proposed for this
regulatory enzyme.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (5K):
[in a new window]
Scheme 1.
In this work, we have cloned the gene for the
-monoglucosyldiacylglycerol synthase from A. laidlawii
membranes and a sequence analog from the pathogen Streptococcus
pneumoniae and propose these genes, on the basis of sequence
similarities, to belong to a new large group of lipid
glycosyltransferases that are widely spread in nature. We also present
a functional comparison between the two cloned glucosyltransferases and
discuss structural properties based on two- and three-dimensional fold
predictions from the primary structure. A striking similarity to two
new, related structures for an Escherichia coli
glycosyltransferase and epimerase, respectively, is indicated.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Strains and Genomic DNA-- Strain A-EF22 of A. laidlawii was cultivated as described by Karlsson et al. (3), and genomic DNA was prepared using the kit GenomicPrepTM (Amersham Pharmacia Biotech). The growth of S. pneumoniae strain 19F CCUG 3030 was performed in Todd-Hewitt medium. For DNA extraction, an overnight culture was harvested, and the pellet was resuspended in H2O and heated at 95 °C for 10 min. The supernatant from the following centrifugation contained the DNA.
PCR Amplification-- The N-terminal sequence of the purified MGlcDAG synthase from A. laidlawii (alMGS) (3) was analyzed through Edman degradation, revealing a 20-residue sequence (4). An internal amino acid sequence of 10 residues was determined as above after proteolytic cleavage of the protein and separation of peptides by reverse phase high pressure liquid chromatography.
Degenerated oligonucleotides with the primary sequence 5'-ATT GGT ATI TT(T/C) TCI GAA GC-3' and 5'-TTT ATC TGG ICC (A/G)TC (G/T)CC-3' (DNA Technology, Denmark) were synthesized and used in a PCR amplification with genomic A. laidlawii DNA as the template. Amplification conditions using AmpliTaq® DNA polymerase were 30 cycles at 96 °C for 1 min, 48 °C for 1 min, and 72 °C for 1 min followed by a final extension at 72 °C for 10 min. Purified PCR product was ligated into a pCR-ScriptTM Amp SK(+) cloning vector (Stratagene) and cloned in TOP10F' cells (Invitrogen). Screening for positive colonies was performed by blue-white color selection, combined with PCR or colony DNA hybridization. Sequence analysis of positive clones was performed using either vector or gene-specific primers and ABI PRISM® BigDyeTM Sequencing Kit (PE Applied Biosystems).
Southern Blot and Hybridization--
Restriction endonuclease
HindIII was used for a complete digestion of A. laidlawii genomic DNA. The DNA fragments were separated by agarose
gel electrophoresis and transferred to a Hybond-N membrane in a
Southern blot procedure (5). Probes amplified by PCR with gene-specific
primers from the cloned fragment of the Almgs gene were
[35S]dATP-labeled by nick translation and used in DNA
hybridization for 18 h at 50 °C. DNA fragments with the
molecular mass corresponding to the hybridization bands were purified
and used in a ligation reaction with the pCR-ScriptTM Amp
SK(+) vector linearized by HindIII. Hybond-N membrane,
[-35S]dATP and the nick translation kit N5500 were all
purchased from Amersham Pharmacia Biotech. The hybridization was
visualized by electronic autoradiography (Packard Instant
ImagerTM).
Cloning and Expression-- Oligonucleotides were designed for the start and stop codon region for the MGS gene from S. pneumoniae (SPmgs), with the forward primer structure 5'-AAA GTG AGG TAA TCT ATG CGA ATT G-3' and reversed primer sequence 5'-GCT GTT CCT CTT TCT ATT CTT CAT-3'. The corresponding oligonucleotides for the MGS gene from A. laidlawii (ALmgs) were designed with the sequence 5'-AAA GTG AGG TAA TCT ATG AGA ATT GGT ATT TTT TCG G-3' and 5'-CTA CTT TTT ATT CAA TTT TTT GTT ATT TTT ATC-3'. Genomic DNA was used for PCR amplification, and the products were ligated into the pCR-Script vector and cloned as described above.
The alMGS was also constructed with an N-terminal His6
tag,2 using the E. coli strain BL21 (Novagen) for cloning of the pET15b recombinant.
Both TOP10F' and BL21 were grown on agar plates supplemented with 100 µg/ml carbenicillin. Protein expression of all recombinant strains
was performed in 1× LB medium supplemented with 50 µg of
carbenicillin/ml. The strains were grown at 37 °C, and 1 mM isopropyl-1-thio--D-galactopyranoside was
added at A600 = 0.6. Cells were harvested
by centrifugation after 5 h of incubation.
Amphiphiles Used-- Synthetic rac-1,2-dioleoyldiacylglycerol was purchased from Sigma. Synthetic 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphoglycerol, 1,2-dioleoyl-sn-glycero-3-(phospho-L-serine), and beef heart cardiolipin (CL) were purchased from Avanti Polar Lipids (Alabaster, AL). The MGlcDAG was prepared as described by Karlsson et al. (3). Radiolabeled MGlcDAG, synthesized in a standard assay procedure with the cloned MGlcDAG synthase, was extracted from the TLC plate (Silica 60) with chloroform/methanol, 2:1 (v/v). Dodecylphosphate-rac-glycerol (PGD) was from Alexis Corp., and CHAPS detergent was from Roche Molecular Biochemicals.
Solubilization of Cells and Lipids-- E. coli cells were solubilized in assay buffer (110 mM HEPES, pH 8.0, 22 mM MgCl2, and 22 mM CHAPS), by extensive vortexing three times during incubation on ice for 3 h. The protein concentrations in solubilized cell extracts were between 4-5 mg/ml and determined by a micro-BCA kit (Pierce). Mixed micellar dispersions were prepared as described by Karlsson et al. (3), with an exception for the assay buffer (cf. above).
Enzymatic Assays-- In the standard assay for MGlcDAG synthesis, 25 µl of protein solution (see above) was added to 20 µl of lipid micellar solution and incubated on ice for 30 min. The reaction was started by the addition of 5 µl of UDP-[14C]glucose to give a final concentration of 1 mM (30 GBq/mol). Standard lipid concentration was 10 mM (1 mM DAG substrate in addition to the activator 1,2-dioleoyl-sn-glycero-3-phosphoglycerol). After 30 min of incubation at 28 °C, the reaction was stopped with 375 µl of methanol/chloroform, 2:1 (v/v), and the lipids were extracted and separated by TLC (2). Radiolabeled enzymatic product from MGlcDAG synthesis in vivo was utilized in a DGlcDAG synthesis assay (7), using a nearly homogenous fraction of the DGlcDAG synthase from A. laidlawii. The lipid products on the TLC plates were visualized and quantified by electronic autoradiography (Packard Instant ImagerTM). All assays were done in duplicate.
The glucolipid products were also identified with a spray reagent. Lipid extracts were first separated by TLC as above, sprayed with a sulfuric acid/methanol mixture 1:1 (v/v), and exposed to 170 °C. Lipids containing sugar moieties were colored purple after ~2 min (8).
Growth of Recombinant Strains--
Overnight cultures were grown
in 1× LB medium (50 µg of carbenicillin/ml) and used for inoculation
(2%) to the same medium containing 14.8 kBq of
[14C]acetate/ml. The strains were grown at 37 °C, and
2 mM isopropyl-1-thio--D-galactopyranoside was added at A600 = 0.6. Cells were harvested by
centrifugation after 4 h of incubation. Lipids were extracted from
the cell pellet twice by chloroform/methanol (2:1 and 0:1, v/v) and
separated by TLC (0.2-mm Silica Gel 60) developed in
chloroform/methanol/acetic acid (65:25:10, v/v/v). Plates were
visualized by autoradiography (cf. above). The radioactivity
is incorporated into the lipid acyl chains and was assumed to be
approximately equal except for cardiolipin (4 chains), which was
predicted to be labeled twice. A similar in vivo
labeling was also performed with addition of [14C]UDP-Glc
instead of acetate.
Sequence Analysis and Structure Prediction-- The amino acid sequence of the alMGS was used in searches for homologous/analogous sequences with PSI-BLAST (9) at the NCBI, in the data base of finished and unfinished genomes, and the Conserved Domain Data base at NCBI. Preliminary sequence data were obtained from the Institute for Genomic Research through their Web Site. Genes listed in Table II and Fig. 1 are selections from searches updated March 6, 2001. Prediction of the primary and secondary structures of the MGS sequences were performed with tools available at the ExPASy Molecular Biology server (Swiss Institute of Bioinformatics) and with the Wisconsin Package version 9.1 (Genetics Computer Group, Madison, WI). For the three-dimensional structure predictions, the three-dimensional fold recognition service at the EMBL Web site (10) and Protein Data Bank (PDB)-ISL (11) at SCOP were utilized.
Nucleotide Sequence Accession Number--
Nucleotide sequence
data have been deposited at GenBankTM with accession number
AF349769.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Gene Cloning and Sequence--
From the purified MGlcDAG synthase
of A. laidlawii (3), the N-terminal (MRIGIFSEAYLPLISGVV) and
an internal (FIIIGDGPDK) amino acid sequence was determined. A PCR
amplification from chromosomal DNA with the corresponding degenerated
DNA oligonucleotide primers yielded a 762-base-long nucleotide
fragment. This was used as a base for probes in a Southern blot
procedure and identified a 4.2-kilobase-long nucleotide
sequence described in Table I. An open
reading frame of 1197 bases, a putative transcription start at base
11, and a potential ribosome-binding site were identified on the
basis of a matching of the translated sequence with the two amino acid
sequences above. The G + C content of the DNA was typical for
acholeplasmas (30.8%). This open reading frame was coding for a
protein with 398 amino acids, lacking a signal peptide (according to
SignalP) and given the gene name ALmgs. The amino acid
sequence is not related to the ones in the UGT Nomenclature Committee
(12). No other potential lipid-synthesizing enzyme genes were present
on the contig. However, the two tRNA-amino acid synthases indicate a
conserved chromosomal environment. The A. laidlawii
tRNALeu has been described earlier in Ref. 13.
|
The amino acid sequence for ALmgs was used as a query in a
PSI-BLAST homology search. A selection of the best hits are presented in Table II; among the 20 top ones
putative glycosyltransferases from a large variety of organisms were
found. All of these belong to family 4 (retaining GTs) in the
glycosyltransferase systematics (CAZy) (14) and share the
typical residues for -GTs in this class and belong to family D in
the classification by Breton et al. (15). However, the best
scores were revealed in a data base for finished and unfinished
microbial genomes. Here, the best hit was a sequence coding for a
protein in Treponema denticola with 36% amino acid identity
to the MGlcDAG synthase (the six best hits all had identities above
30%). The translated sequences from Enterococcus faecalis,
Streptococcus pyogenes, S. pneumoniae, and
T. denticola (second row) were aligned
with the alMGS sequence (top) (Fig.
1). The conserved residues in all five
sequences were mainly focused to three domains: the first 40 residues,
residues 90-130, and above all amino acids 280-310, which contains
the characteristic motif EX7E of family 4 GTs
(cf. above).
|
|
A search in the protein domain data base (at NCBI) showed that alMGS and the potential homologs belonged to pfam00534, which is the glycosyltransferase group 1. These proteins transfer NDP-linked sugars, like glucose, galactose, mannose, and X-glucose, to a variety of acceptor substrates such as glycogen and lipopolysaccharides. Part of the S. pneumoniae gene (Fig. 1) has been annotated before but with unknown function (16); the full sequence was retrieved from a contig (gnl|TIGR|S.pneumoniae_3836) in the finished and unfinished microbial genome data base and was given the name SPmgs. In order to establish the potential functions of the proteins in Fig. 1, SPmgs, ranked fifth in the figure, was PCR-cloned from S. pneumoniae chromosomal DNA.
Gene Functions--
The genes ALmgs and
SPmgs were placed under control of the lac
promoter but out of frame with the -galactosidase gene in the
pCR-Script vector in an E. coli TOP10F'. A putative
ribosome-binding site found at
10 upstream from the start codon in
SPmgs was used for both constructs. The ALmgs
gene was also ligated into a pET15b vector and overexpressed in
E. coli BL21 as a recombinant protein variant with a
His6 tag fused to the N terminus.2
Harvested cells from induced recombinant and control strains were
solubilized by CHAPS detergent, and standard in vitro assays for MGlcDAG synthesis were performed, using substrates and activator lipid according to experimental procedures (cf. Ref. 3). The results (Table III) revealed that the
encoded proteins were able to catalyze the assumed or predicted
glucosylation reaction. The same radiolabeled product was synthesized
independent of which of the two substrates, [14C]UDP-Glc
or [14C]DAG, was labeled. In order to verify that the
lipid product synthesized in vivo (Fig.
2, lane 5) was
MGlcDAG, it was extracted from the TLC plate and used in an in
vitro assay for DGlcDAG synthesis with purified DGlcDAG synthase
from A. laidlawii membranes (7). This enzyme can only use
-MGlcDAG as the lipid substrate and not
- MGlcDAG or Gal
variants. The extracted, radiolabeled lipid (Fig. 2, lane
1), could indeed be used as substrate in the DGlcDAG synthesis (Fig. 2). The intensity of the radiolabeled product was
increased when 14C-labeled UDP-glucose was used
(lane 3). The MGlcDAG TLC spot was also
identified as a glycolipid by charing with sulfuric acid/methanol (1:1,
v/v) (see "Experimental Procedures"). A typical purple color characteristic for glycolipids was observed (data not shown).
|
|
Hence, the two genes ALmgs and SPmgs, with the translated amino acid sequences indicated in Fig. 1, encode analogous enzymes, which both perform the synthesis of the membrane lipid MGlcDAG.
Lipid Composition in Recombinant Cells-- The lipid composition in the recombinant E. coli strains was analyzed by incorporation of [14C]acetate into the lipids during growth. Four major lipids were recognized on the TLC plates: the glucolipid MGlcDAG, phosphatidylethanolamine (PE), and the negatively charged lipids phosphatidylglycerol (PG) and CL (Fig. 2). The control TOP10F' strain, containing a pCR-Script vector, had a lipid composition normal for E. coli wild type, with about 72% PE and 28% negatively charged lipids (Table III). The recombinant strain with expressed alMGS contained a significant fraction of MGlcDAG, about 10%. The fraction of anionic lipids was kept constant, while the nonbilayer lipid PE had decreased to about 63%. However, the homologous glucosyltransferase from S. pneumoniae, although active in vitro, did not affect the lipid composition in vivo, and only traces of MGlcDAG were found (Table III, Fig. 2). The BL21 strain, overexpressing the His-tagged alMGS, showed a slightly lower synthesis of the glucolipid compared with TOP10F', indicating that the N-terminal His extension did not seriously effect enzyme activity. The overexpressed GTs decreased the growth rates compared with controls, and the latter reached the stationary phase faster, which may influence the fractions of CL and PG.
Lipid Environment and Enzyme Activity-- The native A. laidlawii MGlcDAG synthase is activated in an essential manner by substantial amounts of negatively charged lipids, especially PG (2, 17). E. coli cells overexpressing the GTs were solubilized and diluted with CHAPS/1,2-dioleoyl-sn-glycero-3-phosphocholine micellar dispersions supplemented with various fractions of negative lipids: DOPG and cardiolipin, due to their occurrence in A. laidlawii and S. pneumoniae membranes (18, 19), and phosphatidylserine (1,2-dioleoyl-sn-glycero-3-(phospho-L-serine)) and PGD because of their PG-like properties.
The results in Fig. 3 show that all four
lipids were potent activators for the two GTs, but to different
extents. An increased fraction of PG and CL gave sigmoidal-like
activation curves for alMGS, while spMGS responded only to PG.
Cardiolipin was able to activate the spMGS but to a very low extent.
The lipid-like detergent, PGD, showed only slightly activating effects
on spMGS. This was also true for the alMGS at lower concentrations, but the activity increased significantly at concentrations above 25%. Without supplemented DAG substrate, traces of MGlcDAG product could
still be detected. This was most probably due to a minor fraction of
endogenous DAG present in the added E. coli cell suspension. The total content of E. coli lipids present in an assay was
estimated to be less than 50 nmol (cf. the supplemented
amounts of 500 nmol). All observed effects on the cloned alMGS (Fig. 3)
were in agreement with earlier studies of the native enzyme (2,
17).
|
Structure Predictions-- alMGS is firmly anchored in the membrane, and detergents are needed for solubilization (3). Potential hydrophobic transmembrane (TM) segments in the sequences were investigated with a number of prediction methods at the ExPASy Molecular Biology server. One TM (residues 3-22) was proposed according to HMMTOP (20), TMpred (residues 3-24) (21), and TopPred2 (residues 4-24) (22), but not by the SOSUI (23). The orientation of the putative TM was uncertain. This segment also had a substantial amphipathic character according to a hydrophobic moment analysis (24). According to the majority of methods used, spMGS was a soluble protein lacking TM segments, except by HMMTOP (20), predicting one at residues 3-21.
The alMGS sequence showed a low homology to proteins in the structure
data base (PDB). However, a three-dimensional fold prediction method
based on homologous sequence searches (10) listed MurG from E. coli (sequence identity of 14%; 26% similar amino acids), which
encodes for a glycosyltransferase catalyzing the last step in the
peptidoglycan precursor pathway (25, 26), and a soluble UDP-N-acetylglucosamine 2-epimerase from E. coli
(27) (12% identical and 24% similar amino acids). These two have very
similar structures (27), but MurG was proposed to be attached to the
inner membrane (no predicted TM). An alignment of MurG and alMGS by
ClustalW was used to find sequence and potential structural
similarities (some gaps included) between the two membrane-associated
proteins. The aligned sequences were marked with the predicted
secondary structure for alMGS (from Jpred (28)) and the established
structure of MurG recently determined (29). The results (Fig.
4) indicated that the alMGS exhibits a
similar but not identical secondary structure and topology to
MurG and the epimerase. Besides this, initial circular dichroism
studies of a purified His-tagged alMGS suggest both - and
-structures,2 in agreement with the
/
open sheet
structure determined for the MurG domains.
|
Furthermore, two regions in this alignment had higher identities (Fig.
4); residues 62-105 and 290-318 (the EX7E motif) in alMGS
showed similarity to residues 71-114 (25% identity) and 254-278
(37% identity) in MurG. A closer study of residues 74-85 in the alMGS
revealed a mixture of hydrophobic and basic amino acids in a predicted
amphipathic -helix (24). The corresponding sequence in MurG
(residues 84-95) is an
-helix proposed to be part of the
membrane-binding domain (29). The amphipathic characters are also
evident from helical wheel presentations (data not shown). Residues
102-106 in MurG consist of a glycine-rich loop (G loop) localized
between a
-strand and an
-helix (29). This motif is related to
the one included in the classical Rossmann fold (30, 31).
Interestingly, a similar motif is present in the alMGS (putative G loop
sequence SXGXXG) (Fig. 4).
Other membrane-binding segments may be present as well. Searching the PDB Intermediate Sequence Library at the SCOP data base (11) with the alMGS sequence as a probe revealed domains in the two related botulinum (PDB structure 3BTA) and tetanus (PDB structure 1A8D) neurotoxins, close to the binding site for the negatively charged neuronal ganglioside lipid (32). Sequence segment 212-260 in alMGS, containing two conserved regions in the potential lipid GTs (Fig. 1), could be modeled on the PDB 1A8D structure template by SwissModel (ExPASy server (33)). Positions 225-239 had the largest hydrophobic moment (24) for the entire alMGS. Likewise, this segment could also be modeled on the membrane-binding, 62-106 segment in MurG (cf. Fig. 4). Another motif spanning from about residue Ile111 to Tyr127, with a large hydrophobic moment in alMGS, was highly conserved in all the potential lipid GTs (Fig. 1). This amino acid stretch was, despite a low similarity, possible to align with a motif (Pro121-Lys136) conserved among MurG proteins (29). Generally, amphipathic segments (24) were less frequent and of smaller magnitude in MurG than in alMGS.
A theoretical pI was calculated to be around 9 (or higher) for the sequences in Fig. 1, with the exception of spMGS and E. faecalis with a pI of about 5-6. For alMGS, a high number of basic residues were found in the N-terminal half, while the second half was dominated by acidic residues. This polarization of charges along the sequence gave a high pI (~10) for the N-terminal halves and a lower pI (~7) for the C-terminal part, a difference that was analogous but lower for spMGS. Interestingly, a similar pattern of charge distribution is valid for MurG with a pI for the full sequence calculated to be 10.2, while the C-terminal had an acidic pI of 6.2.
Hence, the alMGS lipid glucosyltransferase seems to have several
structural features in common with certain membrane-binding proteins of
known structure, especially the E. coli GT MurG.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Lipid Glycosyltransferase Genes--
The genes for the well
studied MGlcDAG synthase from A. laidlawii strain A-EF22 was
cloned, and the encoded catalytic function was confirmed. In the
standard assay procedure, rac-1,2-diacylglycerol was
utilized as the acceptor and UDP--glucose as the donor substrate for
synthesis of the lipid product MGlcDAG. The stereochemistry of the
sugar moiety was characterized indirectly by a coupled enzymatic
synthesis of the subsequent glucolipid DGlcDAG, which specifically
demands
-MGlcDAG as substrate (7).
-MGlcDAG is not present in
A. laidlawii (34). Related Gram-positive bacteria, like the
ones in Fig. 1, all contain the lipid
-MGlcDAG in their membranes
(35).
In addition, -MGlcDAG is the structural base of the lipoteichoic
acid in S. pneumoniae, anchoring this cell wall polymer into
the cytoplasmic membrane (36). The visualization of MGlcDAG from the
S. pneumoniae gene (clone SPmgs; Fig. 2) strongly
indicates that the corresponding genes in Fig. 1, being more similar to the alMGS sequence than spMGS, all encode the MGlcDAG synthesis function. Likewise, treponemas and other spirochetes all contain a
monoglycosyl-DAG, where the hexose is glucose, galactose, or mannose
(37). The T. denticola sequence in Fig. 1 most likely encodes the GT needed for this synthesis. In an analogous manner to
S. pneumoniae, this lipid may also be the anchor to the
complex outer membrane sheath lipid OML521 in T. denticola
(38). Additional homologs from other pathogens, not shown here, were
also found in, for example, Streptococcus mutans,
Clostridium acetobutylicum, and Streptococcus
equi.
In the Gram-positive sequences of Fig. 1, the MGlcDAG synthase genes are adjacent to another gene (potentially in an operon), tentatively identified by us as glycosyltransferases of CAZy family 4 (cf. above). No other GT was identified next to the A. laidlawii MGS gene (see Table I). Likewise, in the related mollicutes Mycoplasma pneumoniae, three potential lipid GT genes lay separated on the chromosome.3 However, the alMGS sequence was not related to the M. pneumoniae ones, in agreement with the different glycolipid structures.3
This potential group of lipid GTs seems to be widely spread in nature
according to the list of selected orthologs in Fig. 1 and Table II.
Prokaryotes, including Gram-positive and Gram-negative eubacteria, and
archaea are represented, but the list also contains sequences from
eukaryotes. Interestingly, they were found also in the
hyperthermophiles Thermotoga maritima and Pyrococcus
horikoshii, members of eubacteria and archaea, respectively. This
all indicates that a common ancestor to these orthologs was developed
very early in the evolution, before the separation of the bacterial
from the archaeal lineages. All analogs (Fig. 1 and Table II) analyzed contained the EX7E motif typical for the retaining -GTs
of CAZy family 4 (14). A number of the analogs in Table II are involved in the synthesis of various lipids or lipid-based molecules. The Borrelia burgdorferi enzyme synthesizes
monogalactosyl-DAG,4 in
accordance with the reported presence of this lipid in B. hermsii (40), and the S. pneumoniae homolog encodes the
-MGlcDAG synthase (this work). Lactococcus,
Deinococcus, Thermotoga, and Pyrococcus species (Table II) all contain various
glycolipids, including
-MGlcDAG in the two former ones (35, 41).
However, the gene from Pseudomonas aeruginosa is not the one
synthesizing the excreted rhamnolipid (42). The
Synechocystis gene, ranked as number 11 (Table II), was
recently suggested as a lipid
-glycosyltransferase catalyzing the
synthesis of sulfoquinovosyl-diacylglycerol (43).
The Bacillus subtilis gene (Table II) is
tuaC involved in lipoteichoic acid synthesis (44).
Number 17 is Rv0557 from Mycobacterium tuberculosis, and it
was recently identified as a mannosyltransferase (PimB) acting on a
phosphatidylinositol lipid (45). In CAZy family 4 (14), there are more
than a dozen open reading frames from Arabidopsis thaliana.
One of these genes (CAB69850; Table II) showed homology to the MGlcDAG
synthase from A. laidlawii and an even higher similarity
(35% identity) to the proposed sulfoquinovosyl-diacylglycerol synthase
from Synechocystis (cf. above). Like the
-MGalDAG synthase (a GT) from cucumber (46), the two analogs from
Arabidopsis and Synechocystis (slr0384) seemed to
contain a leader and signal peptide of about 103 and 31 amino acids,
respectively (ChloroP/SignalP prediction). They are probable transit
peptides required for import through the chloroplast envelope and
export to a proper Synechocystis compartment.
Hence, the alMGS enzyme is member of a potentially large and conserved
group of lipid glycosyltransferases in nature. Most important, this
group is not closely related sequencewise to the corresponding
-MGalDAG synthases in plant chloroplasts (47).
Regulation of Activity--
The enzymatic regulation of the
MGlcDAG synthesis in A. laidlawii has been extensively
characterized (2, 6). Certain lipids activated the alMGS due to
their charge properties, with PG as the most potent activator. Here,
the two enzymatic activities expressed in E. coli were
studied in a mixed micellar system in vitro and with respect
to the effects of negatively charged lipids (Fig. 3). The sigmoidal
curves shown for PG and CL in the activation of alMGS reached their
maximum at a similar fraction of negative charges (two in CL) (Fig.
3B). Similarly, PG also stimulated the activity of spMGS,
while a very poor response was given by CL. This difference in
regulatory properties is very interesting, since PG and CL are major
lipids in S. pneumoniae (19), but only PG has been found in
this strain of A. laidlawii (18). The binding of alMGS to
lipid bilayers was recently shown to be modulated by electrostatic
interactions,2 with a preference for binding to PG- and
CL-enriched membranes. The regulation of spMGS activity may be governed
in a different way; the low response to CL and the stimulatory effects
by PG indicate a reduced ability to interact with CL. spMGS has
substantially different charge properties as illustrated by the lower
pI of its N- and C-terminal halves (domains) (see above). This may
serve to inhibit synthesis of too much nonbilayer-prone lipid, since both CL and MGlcDAG have such properties. Alternatively, CL is a true
inhibitor of the spMGS enzyme. In the A. laidlawii used, lacking CL, this is evidently not the case. In the two ALmgs
recombinant clones in vivo, the new nonbilayer-prone
glucolipid was synthesized to ~10 mol %. The fraction of PE was
lowered to the same extent, while the fraction of the negatively
charged lipids was kept. This down-regulation of the major nonbilayer
E. coli lipid may be an enzymatic regulation of the lipid
synthesis to keep certain biophysical properties, like the spontaneous
curvature (48), intact in the bilayer. However, the nonbilayer-prone
-MGalDAG from cucumber did not cause an analogous reduction of only
PE in E. coli (46).
The lack of in vivo MGlcDAG synthesis in the SPmgs clone may depend on (i) the presence of CL in E. coli (cf. above); (ii) a lower density of basic amino acids in spMGS, leading to a weaker binding to an intracellular anionic membrane; or (iii) an inhibitor acting on the enzyme in vivo but not in vitro. Early studies of this enzyme in S. pneumoniae (49) localized the glucosyltransferase activity to a soluble fraction, indicating that the protein was not tightly bound to the membrane. The major nonbilayer-prone lipid PE in E. coli did not act as an inhibitor to this enzymatic activity according to results from in vitro experiments (data not shown). PE has not been found in S. pneumoniae.
Structure Proposal--
Up to now only a handful
NDP-glycosyltransferases have been structurally determined. The
majority of these are using the inverting mechanism and the
glycosidic bond formed in the products are in the -configuration,
but one exception is the newly determined structure of LgtC in
Neisseria meningitidis (50), which uses a retaining
mechanism. The sequence similarity between these structures is low, and
they are classified into different glycosyltransferase (CAZy) families
(14). However, their three-dimensional structures fall into only two
superfamilies (51). The three-dimensional fold prediction (10) for
alMGS (see "Results") is proposed to be similar to one of
these, containing the membrane-bound E. coli MurG (29) and
soluble UDP-N-acetylglucosamine 2-epimerase (27) but also
phage T4
-GT structures (51). Similar predictions were valid for all
of the most closely related sequences in Fig. 1 and for
Lactococcus lactis and B. burgdorferi in Table
II. Likewise, this was also the case for the cucumber
-MGalDAG GT
and its Arabidopsis homolog (data not shown). The latter two
and MurG belong to CAZy family 28 (14), strongly indicating structural
similarities between the latter and family 4, including alMGS. The LgtC
in N. meningitidis, with a retaining mechanism, belongs to
family 8. Although they have an analogous catalytic mechanism, the LgtC
and alMGS do not show any strong structural homology.
A prediction of alMGS secondary structures and an alignment along the MurG sequence (Fig. 4) revealed several surprising similarities and several regions potentially involved in membrane binding. One of these (positions 212-260; Figs. 1 and 4) could be modeled on an analogous region in two membrane-binding toxins (cf. above), but most typical was the amphipathic character of all of these regions. Such features are described for many proteins binding to lipid bilayer surfaces, and they are analyzed in more detail for a number of established amphipathic helices from the latter (recently reviewed by Johnson and Cornell (52)). Most similar in this collection of amphipathic helices was a membrane binding segment of DnaA (positions 366-388), initiating chromosome replication in E. coli (53); it aligned with positions 218-240 in the region with the largest hydrophobic moment of the entire alMGS sequence (cf. Figs. 1 and 4). The importance of this DnaA segment for phospholipid interaction is visualized by recent mutant studies (54).
Searching the sequences for several lipid-binding proteins, we found
several intriguing similarities. The negatively charged, signal
recognition particle receptor FtsY of E. coli, integrating into anionic phospholipids (55), has a positively charged amphipathic -helix 4 in the structure (56), very similar in sequence to the
position 75-91 amphipathic segment in alMGS (Fig. 4). Likewise, but
with a slightly lower similarity, was the resemblance of this alMGS segment with the C-terminal amphipathic anchor segments of the
LgtC galactosyltransferase (57) and the E. coli
phosphatidylserine synthase (58). Phosphatidylserine synthase is the
rate-keeping step for the synthesis of the major nonbilayer-prone lipid
PE in E. coli (39) and associates to a negatively charged
lipid surface (6). The features discussed above and the similarity of
this amphiphilic, positively charged segment in alMGS with the aligned
membrane-binding segment in MurG (first box in
Fig. 4) strongly support a similar anchoring function for these two. In
the soluble epimerase (above) the corresponding helix sequence segment
has fewer positive and more negative charges and is now the contact
region in the dimer (27), with no membrane attachment.
A model (schematic diagram) for the interaction and anchoring of alMGS
with a lipid bilayer surface by a combination of charge-charge and
hydrophobic interaction is shown in Fig.
5. It is based on (i) the indicated
similarities (see "Results") between the structurally determined
MurG glycosyltransferase and alMGS (and modeled on the former); (ii)
the cooperative dependence of alMGS activity on the activator lipid PG
(3); (iii) the corresponding dependence of alMGS binding to PG-enriched
(and CL-enriched) bilayers2; (iv) the ability to release
most alMGS from membranes only by detergents and chaotropic
agents5; and (v) the presence
of several potential amphipathic helix segments in the alMGS sequence,
typical for many lipid surface-associated proteins (cf.
Johnson and Cornell (52)). Here, a close approach of the active site
region to the bilayer surface, containing the hydrophobic substrate
DAG, may be governed or modulated by the type and amount of negatively
charged activator lipids.
|
In summary, the enzyme synthesizing the major nonbilayer-prone membrane
lipid MGlcDAG in A. laidlawii is related to a large group of
lipid glycosyltransferases in nature. It has homologs in related
pathogenic bacteria and a structure potentially similar to E. coli MurG, and it is probably attached to the membrane by charge-charge and hydrophobic interactions.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Mari Norgren (Clinical Bacteriology, Umeå University), for supplying the S. pneumoniae strain, and we are grateful to Viola Tegman and Kerstin Hjortberg for technical assistance. We are also grateful to Dr. Olof Karlsson and Dr. Susanne Vikström for valuable discussions and assistance. Dr. Bo Ek (SLU, Uppsala), performed the determination of the N-terminal and internal sequence of the alMGS. We thank Eva Selstam (Umeå University) for glycolipid identification with the spray assay procedure. Preliminary sequence data were obtained from the Institute for Genomic Research Web site.
![]() |
FOOTNOTES |
---|
* This work was supported by the Swedish Natural Science Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF349769.
§ These authors contributed equally to this work.
To whom correspondence should be addressed. Tel.:
46-8-16-24-63; Fax: 46-8-15-36-79; E-mail: ake@dbb.su.se.
Published, JBC Papers in Press, April 6, 2001, DOI 10.1074/jbc.M102576200
2 L. Li, O. P. Karlsson, S. Berg, and A. Wieslander, submitted for publication.
3 M. L. Rosén and Å. Wieslander, manuscript in preparation.
4 Berg, S., Ostberg, Y., Bergshöm, S., and Wieslander, Å., manuscript in preparation.
5 O. P. Karlsson and Å. Wieslander, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
MGlcDAG, 1,2-diacyl-3-O-(-D-glucopyranosyl)-sn-glycerol;
alMGS, A. laidlawii MGlcDAG synthase;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
CL, cardiolipin;
1, 2-DAG, 1,2-diacylglycerol;
DGlcDAG, 1,2-diacyl-3-O-[
-D-glucopyranosyl-(1
2)-O-
-D-glucopyranosyl]-sn-glycerol;
GT, glycosyltransferase;
-MGalDAG, 1,2-diacyl-3-O-(
-D-galactopyranosyl)-sn-glycerol;
PE, phosphatidylethanolamine;
PG, phosphatidylglycerol;
PGD, dodecylphosphate-rac-glycerol;
spMGS, S.
pneumoniae MGlcDAG synthase;
PCR, polymerase chain reaction;
MGS, MGlcDAG synthase;
TM, transmembrane;
contig, group of overlapping
clones;
Glc, glucose.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Hjelmstad, R., and Bell, R. (1991) Biochemistry 30, 1731-1740[Medline] [Order article via Infotrieve] |
2. |
Karlsson, O. P.,
Dahlqvist, A.,
and Wieslander, Å.
(1994)
J. Biol. Chem.
269,
23484-23490 |
3. |
Karlsson, O. P.,
Dahlqvist, A.,
Vikstroem, S.,
and Wieslander, A.
(1997)
J. Biol. Chem.
272,
929-936 |
4. | Karlsson, O. P. (1997) Maintenance of Lipid bilayer properties: Key role of two glucolipid synthases in the membranes of Acholeplasma kidlawii. Ph.D. thesis , Department of Biochemistry, Umeå University, Umeå, Sweden |
5. | Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. G., and Struhl, K. (eds) (1992) Short Protocols in Molecular Biology , 2nd Ed. , pp. 2:24-2:26, John Wiley & Sons, Inc., New York |
6. |
Salamon, Z.,
Lindblom, G.,
Rilfors, L.,
Linde, K.,
and Tollin, G.
(2000)
Biophys. J.
78,
1400-1412 |
7. | Vikstroem, S., Li, L., Karlsson, O. P., and Wieslander, Å. (1999) Biochemistry 38, 5511-5520[CrossRef][Medline] [Order article via Infotrieve] |
8. | Gurr, M. I., and James, A. T. (1971) Lipid Biochemistry: An Introduction , pp. 162-164, Chapman and Hall, London |
9. |
Altschul, S. F.,
Madden, T. L.,
Schäffer, A. A.,
Zhang, J.,
Zhang, Z.,
Miller, W.,
and Lipman, D. J.
(1997)
Nucleic Acids Res.
25,
3389-3402 |
10. | Huynen, M., Doerks, T., Eisenhaber, F., Orengo, C., Sunyaev, S., Yuan, Y. P., and Bork, P. (1998) J. Mol. Biol. 280, 323-326[CrossRef][Medline] [Order article via Infotrieve] |
11. | Teichmann, S. A., Chothia, C., Church, G. M., and Park, J. (2000) Bioinformatics 16, 117-124[Abstract] |
12. | Mackenzie, P., Owens, I., Burchell, B., Bock, K., Bairoch, A., Belanger, A., Fournel-Gigleux, S., Green, M., Hum, D., Iyanagi, T., Lancet, D., Louisot, P., Magdalou, J., Chowdhury, J., Ritter, J., Schachter, H., Tephly, T., Tipton, K., and Nebert, D. (1997) Pharmacogenetics 7, 255-269[Medline] [Order article via Infotrieve] |
13. | Tanaka, R., Andachi, Y., and Muto, A. (1991) Nucleic Acids Res. 19, 6787-6792[Abstract] |
14. | Campbell, J., Davies, G., Bulone, V, and Henrissat, B. (1997) Biochem. J. 326, 929-939[Medline] [Order article via Infotrieve] |
15. | Breton, I., Bettler, E., Joziasse, D. H., Geremia, R. A., and Imberty, A. (1998) J. Biochem. (Tokyo) 123, 1000-1009[Abstract] |
16. | Grebe, T., Paik, J., and Hakenbeck, R. (1997) J. Bacteriol. 179, 3342-3349[Abstract] |
17. |
Li, L.,
Karlsson, O. P.,
and Wieslander, A.
(1997)
J. Biol. Chem.
272,
29602-29606 |
18. | Steinick, L., Wieslander, A., Johansson, K., and Liss, A. (1980) J. Bacteriol. 143, 1200-1207[Medline] [Order article via Infotrieve] |
19. | Brundish, D. E., Shaw, N., and Baddiley, J. (1967) Biochem. J. 104, 205-211[Medline] [Order article via Infotrieve] |
20. | Tusnady, G. E., and Simon, I. (1998) J. Mol. Biol. 283, 489-506[CrossRef][Medline] [Order article via Infotrieve] |
21. | Hofmann, K., and Stoffel, W. (1993) Biol. Chem. Hoppe-Seyler 374, 166 |
22. | von Heijne, G. (1992) J. Mol. Biol. 225, 487-494[Medline] [Order article via Infotrieve] |
23. | Hirokawa, T., Boon-Chieng, S., and Mitaku, S. (1998) Comput. Appl. Biosci. 14, 378-379[Abstract] |
24. | Eisenberg, D., Schwarz, E., Komaromy, M., and Wall, R. (1984) J. Mol. Biol. 179, 125-142[Medline] [Order article via Infotrieve] |
25. | Mengin-Lecreulx, D., Texier, L., Rousseau, M., and van Heijenoort, J. (1991) J. Bacteriol. 173, 4625-4636[Medline] [Order article via Infotrieve] |
26. | Bupp, K., and van Heijenoort, J. (1993) J Bacteriol. 175, 1841-1843[Abstract] |
27. | Campbell, R., Mosimann, S., Tanner, M., and Strynadka, N. (2000) Biochemistry 39, 14993-5001[CrossRef][Medline] [Order article via Infotrieve] |
28. | Cuff, J. A., Clamp, M. E., Siddiqui, A. S., Finlay, M., and Barton, G., J. (1998) Bioinformatics 14, 892-893[Abstract] |
29. | Ha, S., Walker, D., Shi, Y., and Walker, S. (2000) Protein Sci. 9, 1045-1052[Abstract] |
30. | Bränden, C., and Tooze, J. (1998) Introduction to Protein Structure , pp. 47-64, Garland Publishing, Inc., New York |
31. | Baker, P., Britton, K., Rice, D., Rob, A., and Stillman, T. (1992) J. Mol. Biol. 228, 662-671[Medline] [Order article via Infotrieve] |
32. | Umland, T., Wingert, L., Swaminathan, S., Furey, W., Schmidt, J., and Sax, M. (1997) Nat. Struct. Biol. 10, 788-792 |
33. | Guex, N., and Peitsch, M. C. (1997) Electrophoresis 18, 2714-2723[Medline] [Order article via Infotrieve] |
34. | Hauksson, J. B., Rilfors, L., Lindblom, G., and Arvidson, G. (1995) Biochim. Biophys. Acta 1258, 1-9[Medline] [Order article via Infotrieve] |
35. | Ratledge, C., and Wilkinson, S. G. (eds) (1988) Microbial Lipids , Vol. 1 , Academic Press, London |
36. | Fischetti, V. A., Novick, R. P., Ferretti, J. J., Portnoy, D. A., and Rood, J. I. (eds) (2000) Gram-positive Pathogens , pp. 191-200, American Society for Microbiology Press, Washington, D. C. |
37. | Livermore, B., and Johnson, R. (1974) J. Bacteriol. 120, 1268-1273[Medline] [Order article via Infotrieve] |
38. |
Schultz, C.,
Wolf, V.,
Lange, R.,
Mertens, E.,
Wecke, J.,
Naumann, D.,
and Zahringer, U.
(1998)
J. Biol. Chem.
273,
15661-15666 |
39. | Saha, S., Nishijima, S., Matsuzaki, H., Shibuya, I., and Matsumoto, K. (1996) Biosci. Biotechnol. Biochem. 60, 111-116[Medline] [Order article via Infotrieve] |
40. | Livermore, B. P., Bey, R. F., and Johnson, R. C. (1978) Infect. Immun. 20, 215-220[Medline] [Order article via Infotrieve] |
41. | Sprott, G. D. (1992) J. Bioenerg. Biomembr. 24, 555-566[Medline] [Order article via Infotrieve] |
42. |
Ochsner, U.,
Fiechter, A.,
and Reiser, J.
(1994)
J. Biol. Chem.
269,
19787-19795 |
43. |
Guler, S.,
Essigmann, B.,
and Benning, C.
(2000)
J. Bacteriol.
182,
543-545 |
44. | Soldo, B., Lazarevic, V., Pagni, M., and Karamata, D. (1999) Mol. Microbiol. 31, 795-805[CrossRef][Medline] [Order article via Infotrieve] |
45. |
Schaeffer, M.,
Khoo, K.,
Besra, G.,
Chatterjee, D.,
Brennan, P.,
Belisle, J.,
and Inamine, J. M.
(1999)
J. Biol. Chem.
274,
31625-31631 |
46. |
Shimojima, M.,
Ohta, H.,
Iwamatsu, A.,
Masuda, T.,
Shioi, Y.,
and Takamiya, K.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
333-337 |
47. |
Miege, C.,
Marechal, E.,
Shimojima, M.,
Awai, K.,
Block, M. A.,
Ohta, H.,
Takamiya, K.,
Douce, R.,
and Joyard, J.
(1999)
Eur. J. Biochem.
265,
990-1001 |
48. |
Morein, S.,
Andersson, A.-S.,
Rilfors, L.,
and Lindblom, G.
(1996)
J. Biol. Chem.
271,
6801-6809 |
49. | Kaufman, B., Kundig, F. D., Distler, J., and Roseman, S. (1965) Biochem. Biophys. Res. Commun. 18, 312-318[Medline] [Order article via Infotrieve] |
50. | Persson, K., Ly, H., Dieckelmann, M., Wakarchuk, W., Withers, S., and Strynadka, N. C. (2001) Nat. Struct. Biol. 8, 166-175[CrossRef][Medline] [Order article via Infotrieve] |
51. | Unligil, U., and Rini, J. M. (2000) Curr. Opin. Struct. Biol. 10, 510-517[CrossRef][Medline] [Order article via Infotrieve] |
52. | Johnson, J. E., and Cornell, R. (1999) Mol. Membr. Biol. 16, 217-235[CrossRef][Medline] [Order article via Infotrieve] |
53. | Garner, J., and Crooke, E. (1996) EMBO J. 15, 3477-3585[Abstract] |
54. |
Zheng, W.,
Li, Z.,
Skarstad, K.,
and Crooke, E.
(2001)
EMBO J.
20,
1164-1172 |
55. |
de Leeuw, E.,
te Kaat, K.,
Moser, C.,
Menestrina, G.,
Demel, R.,
de Kruijff, B.,
Oudega, B.,
Luirink, J.,
and Sinning, I.
(2000)
EMBO J.
19,
531-541 |
56. | Montoya, G., Svensson, C., Luirink, J., and Sinning, I. (1997) Nature 385, 365-368[CrossRef][Medline] [Order article via Infotrieve] |
57. | Wakarchuk, W., Cunningham, A., Watson, D., and Young, N. (1998) Protein Eng. 11, 295-302[Abstract] |
58. |
DeChavigny, A.,
Heacock, P.,
and Dowhan, W.
(1991)
J. Biol. Chem.
266,
5323-5332 |