Department of Microbiology, University of
Guelph, Guelph, Ontario N1G 2W1, Canada
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INTRODUCTION |
Glycosyl transferases (GTs) are one
of the most important classes of enzymes on earth, catalyzing the
formation of a large proportion of the biomass on the planet. These are
highly specific enzymes, but collectively represent a diversity of
substrate and acceptor specificities. They catalyze the formation of a
wide variety of molecules, including glycoproteins, glycolipids,
oligosaccharides, and cell envelope and cell wall polysaccharides of
the Bacteria, Archaea, fungi, plants, and
crustaceans. In addition to their role in the formation of structural
molecules, these enzymes play a role in numerous biological processes,
such as immune recognition, protection of pathogens from host immune
responses, intercellular signaling, and biofilm formation. GTs are of
great interest in biotechnology because of their ubiquitous nature.
Despite their catalytic diversity, GTs are subdivided into only two
main classes, either inverting or retaining enzymes, based on the
stereochemistry of the substrate and product (reviewed in reference
31). While sequence analyses reveal that, in general, GTs
exhibit relatively little identity in their primary structure, it has
become evident that certain sequence motifs can be identified. Based on
this, Campbell et al. (6) used basic local alignment search tool (BLAST) analysis (1) and hydrophobic cluster
analysis (HCA) (13) to further classify 553 GTs into 26 families. These families have since been expanded and can be viewed at
the following: http://afmb.cnrs-mrs.fr/~pedro/CAZY/db.html. The
sequence similarities observed within each family were concluded to be
indicative of conserved tertiary structure, and therefore the
three-dimensional fold within the conserved region is predicted to be
the same among members of a given family. The largest of the 47 current
families is family 2, which comprises GTs from all major groups of
living organisms. GT family 2 (GT2) members are inverting GT enzymes using nucleotide-diphospho 
-linked sugar donors to form 
-linked products. Structural predictions and sequence alignments have revealed
that these proteins are characterized by a region of alternating
-helices and -sheets which form domain A, within which is a
conserved DXD motif (29). Previous studies have revealed that the region C terminal to domain A diverges between at least some
processive and nonprocessive GT2 members (24, 29).
The Salmonella O:54 O antigen has a number of unusual
features. It is the only Salmonella O antigen containing
N-acetylmannosamine (ManNAc), and it is the only known
homopolymeric O antigen in this genus (25). The O:54 O
antigen is also the only lipopolysaccharide (LPS) O antigen currently
known to be synthesized by the synthase-dependent pathway
(34). The O:54-specific GT WbbE (RfbA), the initiating enzyme for the O antigen, belongs to family 2. This enzyme is encoded
by the plasmid-borne O:54 O-antigen biosynthesis genes (22, 23,
24). WbbE is a nonprocessive enzyme which has previously been
shown to catalyze the addition of a single ManNAc (ManpNAc) residue to the undecaprenol (Und)-PP-GlcpNAc acceptor. O:54
polymerization and surface expression occur in a
wbbEF-containing Escherichia coli host in which
the entire O-antigen biosynthesis cluster of the host has been deleted.
WbbF (RfbB) is therefore speculated to processively transfer all
subsequent ManNAc residues in alternating (1
3) and (14)
linkages, while simultaneously extruding the growing chain across
the cytoplasmic membrane (24). WbbF has also been
assigned to family 2 (6) and is one of a number of structurally homologous processive enzymes in this family
(24).
To date, the crystal structures of only four GTs have been published:
the DNA -glucosyltransferase of bacteriophage T4 (33), the GT SpsA of Bacillus subtilis (7), the
catalytic domain of a bovine -galactosyltransferase
(16), and most recently, the MurG GT of E. coli
(19). The first exhibits little sequence similarity to any
other GTs. The bovine -galactosyl transferase has been classified
into family 7 of the GTs, and MurG has been classified into family 28 (http://afmb.cnrs-mrs.fr/~pedro/CAZY/db.html). SpsA has been shown to
belong to the more predominant family 2 (6).
Cocrystallization with UDP revealed that domain A in SpsA (and, by
extension, in other family 2 members) represents the nucleotide binding
fold. Unfortunately, the substrate of SpsA is not known, and while the
crystallization data identified specific amino acid residues
interacting with UDP and Mn2+, and a potential catalytic
residue bound to a free glycerol, the identity of the catalytic residue
remains speculative. Currently, it is believed that inverting GTs
possess a catalytic residue which acts as a general base in a single
nucleophile mechanism (10, 21). This residue is generally
thought to be an aspartate or a glutamate.
The present communication provides further analysis of the WbbE
ManpNAc transferase of Salmonella enterica
serovar Borreze. Using site-directed mutagensis and in vivo activity
assays, we confirm that the DXD motif of domain A does indeed play a
critical role in catalysis in this enzyme, as suggested by the
structural data of Charnock and Davies (7) and predicted
by others (14, 17, 30). We identified a consensus sequence
C terminal to domain A in a subset of monofunctional GT2 members, which
is postulated to represent the catalytic domain. Sequence and
predicted structural divergence within the region C terminal to domain
A suggests a fundamental difference between some members of this family
and a potential basis for further subdivision.
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MATERIALS AND METHODS |
Bacterial strains, growth conditions, and plasmids.
Bacterial strains and plasmids used in this study are listed in Table
1. Bacteria were grown at 37°C in
Luria-Bertani broth supplemented, where appropriate, with antibiotics
(ampicillin, 100 µg/ml; kanamycin, 50 µg/ml). For regulated
overexpression of WbbE and WbbF, 100 mM
isopropyl--D-thiogalactopyranoside (IPTG) was added to
strains containing pET30a(+)-derived constructs (Novagen, Madison,
Wis.) at a final concentration of 0.5 mM, and arabinose was added to
those with pBAD24-derived constructs (18) at a final
concentration of 0.02%.
DNA manipulation and analyses.
Restriction enzyme
digestions, ligations, and electroporations were performed as described
by Sambrook et al. (28) and Binotto et al.
(3). Inserts in pBAD24 (18) and pET30a(+)
(Novagen) were obtained by PCR amplification using standard protocols.
Primers were based on the pWQ799 sequences of wbbE and
wbbF (23) (accession no. L39794) and were
designed to incorporate novel restriction sites for cloning. PCR
fragments were cleaned using the QIAquick DNA purification kit from
QIAGEN Inc. (Chatsworth, Calif.). Plasmids were purified using QIAGEN
spin columns. Nucleotide sequencing and oligonucleotide synthesis were
performed by the Guelph Molecular Supercenter (University of Guelph,
Guelph, Ontario, Canada). DNA sequence data were analyzed using
AssemblyLIGN and MacVector software (International Biotechnologies
Inc., New Haven, Conn.).
Analysis of protein structure.
Secondary structure
predictions were done using HCA software (Doriane Informatique, Le
Chesnay, France) as well as the method described by Garnier et al.
(15). Protein homologies were detected using the Position
Iterated (PSI)-BLAST search (2) for conserved motifs.
Multiple sequence alignments were done using Align
(http://vega.igh.cnrs.fr/bin/align-guess.cgi).
Site-directed mutagenesis.
Codons were altered using a
method based on the QuikChange site-directed mutagenesis kit from
Stratagene (La Jolla, Calif.). Mutagenic primers are listed in Table
2 and were used for mutagenesis of
wbbE in plasmid pWQ835. Mutated DNA was sequenced (both
strands) to confirm a single codon change and ensure no additional
changes were introduced.
Cell fractionation.
Bacterial cultures (50 ml) were grown to
mid-exponential phase, and expression of the wbbE gene was
then induced with IPTG. Expression was allowed to proceed for 2 h.
Cells were then harvested by centrifugation, resuspended in 10 ml of
residual medium, and lysed by ultrasonication. Unbroken cells were
removed by centrifugation, and then the membranes were pelleted by
ultracentrifugation (100,000 × g) for 1 h. An
aliquot of the supernatant was kept as the cytosolic and periplasmic
fraction, and the remainder was discarded. The pellet was then
resuspended in 1 ml of 0.1 M Tris, 10 mM MgCl2, and 2%
(wt/vol) Sarkosyl (N-lauroylsarcosine) in order to
solubilize the inner membrane while precipitating the outer membrane
(12). This solution was shaken for 30 min, after which the
precipitated outer membrane was collected as a pellet by centrifugation
at 10,000 × g for 30 min. The supernatant was
concentrated approximately 20-fold, 10% sodium dodecyl sulfate (SDS)
was added to a final concentration of approximately 7.5% (wt/vol), and
the mixture was incubated at 100°C for 1 h. Any precipitate was
removed by centrifugation at 10,000 × g for 10 min,
and the cytoplasmic membrane-containing supernatant was combined with
SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer.
Analysis of LPS.
LPS samples were prepared using the
whole-cell lysate method of Hitchcock and Brown (20).
Samples were separated using 18.5% polyacrylamide gels
(9) and analyzed either by silver staining (32) or by Western blot analysis after electrotransfer to
BioTrace nitrocellulose membrane (Gelman Sciences, Mississauga,
Ontario, Canada). Blots were probed with absorbed polyclonal rabbit
anti-O:54 antisera. The antisera were raised in New Zealand White
rabbits immunized with formalin-killed whole cells of E. coli DH5(pWQ802) (22). Cross-reacting antibodies
were removed by absorbing the serum with whole cells of E. coli DH5. The second antibody was goat anti-rabbit alkaline
phosphatase-conjugated antibody (Jackson ImmunoResearch Laboratories
Inc., West Grove, Pa.). Detection was achieved using
5-bromo-4-chloro-3-indolylphosphate (BCIP) (Roche Molecular
Biochemicals, Laval, Quebec, Canada) and 4-nitroblue tetrazolium
chloride (Sigma Chemical Co., St. Louis, Mo.).
Western blot analysis of proteins.
His-tagged protein
samples were separated using 12% polyacrylamide gels, and then they
were transferred to Biotrace nitrocellulose membranes. Blots were
probed with QIAexpress anti-His6 antibody (QIAGEN Inc.) followed by goat anti-mouse alkaline
phosphatase-conjugated secondary antibody (Jackson ImmunoResearch
Laboratories Inc.) and detection according to instructions supplied by QIAGEN.
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RESULTS AND DISCUSSION |
Localization of WbbE in the cytoplasmic membrane.
The
ManpNAc O:54 O antigen is produced through the action of two
GTs, WbbE and WbbF (24). The WbbE GT has been identified as the first ManNAc transferase to act, transferring a single sugar to
the GlcNAc-PP-Und carrier lipid. Based on polymerization and surface
expression of O:54 LPS in an E. coli host strain with a
deleted O-antigen biosynthesis cluster, as well as structural similarities with other synthases, WbbF is believed to processively transfer all subsequent ManNAc residues, while simultaneously transporting the growing polymer to the external face of the plasma membrane. The final step is ligation of the polymer to lipid A-core.
Previous studies indicated that WbbF is an integral membrane protein
with four transmembrane domains as well as a periplasmic domain
(24). In contrast, WbbE is predicted to possess only one
potential transmembrane domain. To confirm the cellular localization of
WbbE, cell fractionation was attempted using the pWQ835-containing expression clone. Although a variety of arabinose concentrations were
used for induction, no unique protein was visible in any of the
experimental samples, compared to the E. coli DH5(pBAD24) negative control (data not shown). Cells expressing WbbE fused to an
N-terminal His6 tag, encoded by plasmid pWQ839, were
therefore used in cell fractionation studies to visualize the enzyme.
Probing Western blots with an anti-His antibody revealed reactive bands in the inner membrane fraction of the O:54+ strain but not
in the cytoplasmic fraction (Fig. 1). No
protein was detected in the E. coli BL21/pET30a(+) negative
control. The biggest protein in the WbbE-containing lane had an
apparent molecular mass of approximately 25 kDa, smaller than the mass
of 35.5 kDa predicted from sequence data for the His-tagged protein.
The anomalous electrophoretic mobility of the enzyme is a common
observation with membrane proteins, which tend to bind excess SDS and
therefore display a higher electrophoretic mobility than predicted from sequence analysis (4). Other, faster migrating bands were
also detected, suggesting proteolytic degradation. Once the cellular location of wild-type WbbE was determined, the various mutants employed
in this study were assessed in the same manner. All of the mutant
proteins displayed similar banding patterns and were produced at levels
equivalent to that of the wild type (data not shown).
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FIG. 1.
Western blot analysis of His-tagged WbbE in cytoplasmic
and inner membrane fractions. Lanes: 1, cytoplasmic fraction from
negative control BL21/pET30a(+); 2, cytoplasmic membrane fraction of
negative control; 3, cytoplasmic fraction of BL21(pWQ839); 4, inner
membrane fraction of BL21(pWQ839). Fusion proteins were detected with
anti-His antibody.
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To confirm that the WbbE fusion protein was functional, despite the
presence of the His6 tag fusion and the anomalous
electrophoretic mobility, E. coli BL21(pWQ839) was induced
with IPTG and whole-cell lysates were prepared and analyzed by
SDS-PAGE. The silver-stained gel revealed a modification to the core
(data not shown), indicative of a core-plus-two-glycose size increase
as shown previously for a K-12 strain harboring an active WbbE
(24). This band has previously been shown to result from a
single WbbE-specific ManNAc addition to the Und-PP-GlcNAc acceptor. The
host Wzx (the so-called "flippase") exports the lipid-linked
disaccharide to the periplasmic face of the inner membrane
(11), where it is ligated to lipid A-core by the WaaL
ligase. The WbbE GT was therefore functional and localized in the
cytoplasmic membrane, presumably forming a complex with WecA, the
enzyme responsible for the initiating transfer of GlcNAc-1-P to Und-P
(22, 27), and with WbbF.
Analysis of the catalytic role of the conserved aspartate residues
in domain A of WbbE.
Using HCA, Saxena et al. first identified a
region of homology, termed domain A, present in a number of inverting
GTs of known or predicted function (29). Previous analyses
identified the same domain in both WbbE (Fig.
2) and WbbF (24). Possession of this domain is the basis for inclusion in GT2 (6). SpsA is the only member of family 2 to be crystallized (7). The structural data revealed that SpsA is a metalloenzyme, and
crystallization with UDP indicated that domain A is in fact a
nucleotide-binding fold. Within this domain are a number of invariant
aspartate residues. These have been shown to play a critical role in
the catalysis of SpsA, based on cocrystallization with UDP and
Mn2+ (7) and in other GT2 members, based on
mutagenesis (cellulose synthase [30], KfiC [17], ExoM [14]). To
confirm that these residues are also important in WbbE transferase
activity, they were individually converted to alanines. To assess
activity, LPS was prepared from arabinose-induced E. coli DH5(pWQ835) cells expressing either the wild-type
WbbE-WbbF or mutant pWQ835 derivatives. The ability of the mutant WbbE
enzymes to participate with wild-type WbbF in O:54 O-antigen
biosynthesis was assessed by SDS-PAGE and immunoblotting using
polyclonal anti-O:54 antisera (Fig. 3).
In this system, the host WecA function initiates polymerization, and
then WbbE and WbbF complete the polymer and transport it to the
periplasmic face of the cytoplasmic membrane, where it is ligated to
lipid A-core and exported to the cell surface. All LPS preparations were made from the same number of cells; therefore, any change in the
amount of reactive material must have arisen from either a change in
the activity of the mutant WbbE enzyme or in the amount of the enzyme.
Western blot analysis of the corresponding His6-tagged WbbE
derivatives confirmed that there was no significant difference in the
amounts of enzyme synthesized (data not shown). Asp41 in WbbE is
analogous to Asp39 in SpsA, based on its position within domain A at
the end of strand 2 (Fig. 2) (7, 24). Crystallization data revealed that this residue binds N-3 of the uracil base of UDP in
SpsA. In WbbE, however, D41A had no obvious effect on the amount of
O:54 O antigen synthesized. This is potentially due to a greater
opportunity for interactions stabilizing the UDP-ManNAc substrate than
would occur with UDP alone. Additionally, in SpsA, UDP has been shown
to bind through aromatic stacking interactions as well as multiple
hydrogen bonds. Therefore, eliminating one critical residue may not
necessarily result in loss of substrate binding. In WbbE in particular,
the equivalent conserved Asp does not appear to be critical for
activity. These results are in contrast to those obtained for the
analogous replacement in ExoM (14). In both in vitro and
in vivo assays with the ExoM D44A mutant, activity was completely
abrogated. The reason(s) for this discrepancy between the enzymes is
unclear but may reflect the relative cumulative strengths of the
substrate binding interactions.
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FIG. 2.
HCA alignment of WbbE and SpsA. The HCA program writes
protein sequences on a duplicated -helical net and encloses clusters
of hydrophobic amino acids in boxes. The plots are then visually
compared for similarity in the hydrophobic cluster patterns, limiting
analysis to the predicted globular portions of the proteins. Plots are
aligned using traditional sequence alignments as a starting point.
Hydrophobic clusters with obvious similarities were used as anchors for
the structural alignment, as were regions containing Gly ( ) and Pro
( ), which are often present in loops (13). Vertical
lines were drawn to illustrate structurally conserved features. The
prediction of -strands and -helices is based on the observed
correlation between cluster shape and secondary structure
(13). The single-letter code denotes amino acids except
for proline, glycine, serine ( ), and threonine ( ). Conserved
residues are circled.
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FIG. 3.
Analysis of the effect of replacements in domain A of
WbbE on WbbE activity and O:54 LPS expression. Immunoblot of anti-O:54
reactive LPS from E. coli DH5 harboring wild-type or
mutated WbbE and wild-type WbbF. Lanes: 1, pET30a(+) negative control;
2, pWQ835; 3, pWQ835-41; 4, pWQ835-93; 5, pWQ835-93, pWQ818; 6, pWQ835-95; 7, pWQ835-95, pWQ818; 8, pWQ835-96; 9, pWQ835-96, pWQ818.
Polyclonal anti-O:54 antisera were adsorbed against E. coli
DH5, and therefore the R-LPS (lipid A-core) of the recombinants was
not detected. The migration of S-LPS is shown.
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The other invariant Asp in SpsA is Asp99, at the end of the 4 strand
of domain A (Fig. 1) (7). This residue is associated with
a conserved motif found in a number of nucleotide-binding GTs of
various families (5, 10, 35). For example, in family 7, the motif occurs as DXD (16). In GT2 members, the motif
may also occur as DXD; however, the exact number of Asp residues
appears to vary between two and four (7). In WbbE, the
sequence is DXDD (Fig. 2). Changing each Asp individually to Ala and
analyzing the LPS profiles of the mutant strains revealed that the D93A (the first residue of the motif) and D96A replacements completely abrogated WbbE activity, while D95A led to significantly reduced activity (Fig. 3). All three mutant strains could be rescued to normal
activity levels by the presence of the wild-type wbbE gene, confirming that the defect in synthesis was confined to the mutated wbbE gene product. The mutated wbbE genes were
subcloned from the pWQ835 derivatives into the expression vector
pET30a(+) to generate pWQ839-x. Cytoplasmic membrane extracts examined
by Western blot analysis with anti-His antibody revealed no observable
differences in mobilities or protein levels between the wild type and
the mutant fusion (data not shown). The mutagenesis data therefore indicate that Asp95 of WbbE can be converted to a smaller, uncharged Ala with a reduction but not abrogation of activity. This suggests that
Asp95 is unlikely to be the residue which coordinates the Mn2+ ion in WbbE. Alternative candidates are Asp93 and
Asp96, both of which are critical for WbbE activity. It is clear from
these results that sequence alignment in the absence of mutagenesis is
not necessarily enough to identify critical residues even when comparing two GTs from the same family.
Identification of the ED(Y) motif in a subset of GT2 members.
Our previous studies using conventional BLAST searches identified a
region of sequence conservation C terminal to domain A in a number of
nonprocessive GTs (Fig. 2). This domain, domain C, has a complex
secondary structure with a conserved ED(Y) motif in region iv
(24). To determine the relative distribution of this
domain in GT2 enzymes, a PSI-BLAST search was done using the WbbE
sequence and the resulting sequences were scanned for the ED(Y) motif.
All of the sequences identified aligned minimally through domain A;
most have been officially assigned to the GT2 family
(http: //afmb.cnrs-mrs.fr/~pedro/CAZY/db.html). While a number
of sequences aligned only through domain A, 22 also possessed the ED(Y)
motif (more than were identified by the conventional BLAST search). An
alignment of all of the proteins identified by the database search
using WbbE as the query sequence is shown in the top cluster of
sequences in Fig. 4, along with a consensus. The sequences displayed
belong to a broad spectrum of bacterial species from both the
Bacteria and the Archaea (Pyrococcus
abysii, Pyrococcus horikoshii). Interestingly, SpsA was not one of
the proteins identified by the database search. An examination of the
SpsA sequence, plus those of two of its homologues (identified by a
BLAST search using the SpsA sequence), reveals that the proteins agree
with the consensus for domain A (Fig. 4A)
but show little to no sequence conservation through domain C (Fig. 4B).
The ExoM sequence is included in the sequence alignment of Fig. 4
despite the fact that it too failed to be identified by either a BLAST or a PSI-BLAST database search. The reason for including this sequence
is that it possesses an EDT motif in the position equivalent to that of
EDY, and the results of mutagenesis studies suggest that this region
contains the catalytic residue (14; see below). Interestingly, ExoM shows little homology to the consensus, even within
domain A, with the exception of the conserved Asp residues.
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FIG. 4.
Multiple-sequence alignment of proteins with homology to
domains A and C of WbbE. Sequences in the upper cluster were identified
by PSI-BLAST. All sequences were aligned using the Align algorithm
(http://vega.igh.cnrs.fr/bin/align-guess.cgi). Partial sequences are
shown, along with starting positions in parentheses after the protein
names. The lengths of intervening sequences between aligned regions are
indicated in parentheses, whereas gaps within the motif are indicated
by dashes. Amino acids conserved in more than 50% of the proteins are
indicated in bold and in the consensus sequence. Asterisks indicate
strictly conserved residues. The origins and protein accession numbers
for the various enzymes are indicated in order: Se, S. enterica serovar Borreze (accession no. AAC98401.1); Ec, E. coli O7 (AAC27537.1); Se, S. enterica (AF279620.1); Ea,
Erwinia amylovora (Q46635); Hi, Haemophilus
influenzae (C64175); Aa, Actinobacillus
actinomycetemcomitans (BAA28129.1); Ss, Streptococcus
suis (AAF18950.1); Nm, Neisseria meningitidis
(CAB83816.1); Ns, Neissera subflava (AF240672.1); Ng,
Neisseria gonorrhoeae (AAF14359); Ec, E. coli
(AAD50485); Li, Leptospira interrogans (AAD52184.1); Lb,
Leptospira borgpetersenii (AAD12967.1); Vc, Vibrio
cholerae (BAA33634); Pa, Pyrococcus abyssi (G75005);
S.m., Sinorhizobium (Rhizobium)
meliloti (P33700); Sm, S. meliloti (P33697); Bj,
Bradyrhizobium japonicum (AAC04822); Ph, P. horikoshii (A71157): Ye, Yersinia enterocolitica O:8
(AAC60770); Mt, Mycobacterium tuberculosis (CAB06459.1); Ah,
Aeromonas hydrophila (AF146607.1); Sm, S. meliloti (P33695); Bs, B. subtilis (P39621); Bs,
B. subtilis (CAB15817); Bh, Bacillus halodurans
(BAB07092). Shown are domain A (A) and the region C terminal to domain
A (B).
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While the overall similarity is low among the 22 ED(Y)-containing
sequences, there are a number of conserved residues. In addition, all
but four of the sequences also possess a tyrosine after Glu-Asp. Given
the identification of N-terminal domain A as the nucleotide-binding
fold, one can speculate that the region C terminal to this would
logically represent the catalytic domain. Due to the association of
domain C with the ED(Y) motif, the 22 ED(Y)-containing proteins
identified here might be expected to possess a conserved
three-dimensional fold over this region. Possession of a common
structure C terminal to domain A has already been suggested for a
number of processive GT2 members. These proteins possess a region
designated domain B, and this domain is speculated to possess the
catalytic residue(s) (10, 24, 29). Perhaps not
surprisingly, none of the known processive GT2 members aligned through
the ED(Y) region with WbbE.
Recently, Garinot-Schneider et al. reported the identification of a
putative catalytic residue within the EDT motif of ExoM (14) (Fig. 4B). In their study, Asp 187 of the EDT
sequence was replaced with Glu, and subsequent in vivo and in vitro
assays revealed a complete loss of activity. The analogous E186D
mutation was not reported. The authors also reported that the D187 of
ExoM aligned with D191 of SpsA, the residue speculated to represent the
general base based on cocrystallization with glycerol (7). Despite an extremely low level of overall similarity, alignment of the
SpsA sequence with those containing ED(Y) by using the Align algorithm
(http://vega.igh.cnrs.fr/bin/align-guess.cgi) indicates that this
residue also aligns with the Asp of the ED(Y) motif. The evidence
therefore suggests that a catalytic residue may occur at the equivalent
position in ExoM and SpsA and that the ED(Y) motif may also contain a
catalytic base. The identification of a consensus in the
ED(Y)-containing GT2 members suggests, however, that some GT2 enzymes
diverge at least in sequence within the catalytic domain.
Analysis of the catalytic role of the conserved ED(Y) motif in
domain C of WbbE.
To pursue the possibility that ED(Y) contains a
catalytic residue, site-directed replacement was performed on the
strictly conserved Asp and Glu of the motif in WbbE. The ability of the mutant proteins to participate with WbbF in O:54 O-antigen synthesis was assessed (Fig. 5). All LPS
preparations were made from the same number of cells. Analysis of the
data revealed that the conservative change, E180D, resulted in a
significant reduction in O-antigen synthesis, reflecting its effect on
enzyme activity. This suggested that the size of the acidic residue is
important. When the same residue was changed to the uncharged
derivative (E180Q), activity was completely abrogated. Therefore, the
charge on the residue is even more important. Interestingly, in
contrast to the E180D replacement, in E. coli DH5 cells
expressing wild-type WbbE in addition to the E180Q mutant enzyme,
rescue of the mutant phenotype was not complete. Whether this is a true
dominant-negative phenotype remains to be determined.
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FIG. 5.
Analysis of the effect of replacements in the ED(Y)
motif of WbbE on O:54 LPS expression. Immunoblot of anti-O:54 reactive
LPS from E. coli DH5 harboring wild-type or mutated WbbE
and wild-type WbbF. Lanes: 1, pET30a(+) (negative control); 2, pWQ835; 3, pWQ835-180; 4, pWQ835-180, pWQ818; 5, pWQ835-180Q; 6, pWQ835-180Q, pWQ818; 7, pWQ835-181; 8, pWQ835-181A; 9, pWQ835-181A, pWQ818. R-LPS was not detected due to the use of
antisera adsorbed against E. coli DH5. The migration of
S-LPS is indicated.
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When the contribution of D181 was assessed, the conservative
replacement D181E had no effect, while the D181A replacement again
abolished enzymatic activity. O-antigen levels were restored to that of
the wild type in the presence of a functional WbbE, confirming that the
reduction in O-antigen levels was due to the change in WbbE only. D181
is therefore required for activity; however, the size of the acidic
residue at this position is not critical.
Manipulation of the ED(Y) motif indicated that the Glu and the Asp are
both required for enzymatic activity. While only the acidic nature of
D181 appeared to be important, both the size and charge of E180 were
critical for activity. D181 may therefore contribute to the overall
charge at that site. The observations for E180, in contrast, were those
expected of a catalytic amino acid. Among the inverting glycoside
hydrolases, the active site nucleophile may be either a Glu or an Asp,
and mutagenesis indicates that both the size and the charge are
critical for activity. In particular, withdrawal of even 1 Å may
result in a virtually complete loss of activity in vitro
(26). Although loss of activity of the E180D WbbE mutant
was incomplete, one cannot directly compare glycoside hydrolases and
GTs, as they are believed to use different enzymatic mechanisms
(8, 10). While the geometrical position of the general
base in a GT is obviously important, one can speculate that with a
single catalytic residue instead of the two seen in glycoside
hydrolases, the former may exhibit a greater degree of flexibility. The
observation of incomplete loss of activity for E180D is in contrast to
that of Garinot-Schneider et al. for the analogous replacement in ExoM
(14). The reason for this is not apparent; however, the
enzymes possess very little sequence homology over their entire
lengths, and these discrepancies may simply be a reflection of this. In
this context, it is interesting that similar discrepancies were
observed between the two systems with the D-to-A replacement of the
2 Asp of domain A (14).
Catalytic residues are generally found on turns where they can move
into position after the substrate and donor have entered the catalytic
pocket or cleft. The hypothesis that E180 of WbbE may be the catalytic
residue therefore has to be correlated with the predicted secondary
structure of WbbE. Analyses of the WbbE sequence using HCA (Fig. 1) and
the method of Garnier et al. (15) both predict that E180
is present on a turn.
Structural predictions for GT2 members.
While the ED(Y) motif
is proposed to contain the catalytic residue in WbbE, and it appears to
align with the proposed catalytic residues of ExoM and SpsA, it is E180
of WbbE and not D181 which is speculated to represent the catalytic
residue. Based on sequence alignment alone, this would appear to be
displaced by one residue from the equivalent ExoM and SpsA residues
(Fig. 4B). However, results of the DXD mutagenesis, and the general
observation that this motif can vary with respect to the number of Asp
residues associated with it, suggest that sequence alignment is not
enough to predict function. It is possible that E180 is in the same
geometric position as the putative catalytic residues of ExoM and SpsA. Given that SpsA is the only GT2 member to be crystallized, the obvious
question is whether, despite sequence divergence, the ED(Y)-containing
members possess the same three-dimensional fold. HCA was therefore
performed on the protein, and the plot was compared to that of WbbE
(Fig. 1). While there may be some similarity, the data are equivocal.
Based on the combined evidence, the ED(Y) motif is proposed to contain
the general base involved in catalysis in a subset of GT2 members. In
the O:54 biosynthesis system, WbbE and WbbF are both ManNAc
transferases and both belong to family 2. However, they differ
significantly in their enzymatic activities, as the former is
monofunctional whereas the latter is processive. Sequence and HCA
comparisons of the two proteins revealed that the sequence conservation
seen in domain A does not extend to the C-terminal region
(24). The presence of domain C appears to correlate with a
single glycosyl transfer activity (24), whereas at least
some processive members appear to possess a distinctive domain B
downstream of domain A (24, 29, 30). Although the
mechanism of action of processive enzymes is the topic of much debate
(10, 29), one can speculate that such an enzyme would
possess a catalytic domain different from that of a monofunctional
enzyme (i.e., domain B versus C). Combined with the observed sequence
divergence between the ED(Y)-containing GTs and other family 2 members,
it therefore appears that the GT2 enzymes, grouped together by virtue
of their common N-terminal nucleotide-binding fold, are heterogeneous
through the C-terminal region. This situation is reminiscent of the
glycoside hydrolases. These enzymes have chimeric structures,
possessing various combinations of substrate binding and catalytic
domains (8). They are subdivided into families based on
sequence conservation and into clans based on possession of a common
catalytic domain structure. The strong homology within domain A of GT2
members, combined with homologies of subsets over the C-terminal region of these enzymes (domains C versus B), suggests that a similar subdivision may be necessary for this family of GTs.
We thank Paul Amor for preparation of the anti-O:54 polyclonal
antiserum and Sonia Bardy and Corin Forrester for technical assistance.
We also thank the reviewers for their helpful comments.
This work was supported by grants to C.W. and A.J.C. from the Natural
Sciences and Engineering Research Council (NSERC).
After acceptance of the manuscript, two reports of GT crystal
structures were published. L. C. Pederson et al. (J. Biol. Chem. 275:34580-34585, 2000) reported crystallization of a GT2 member, and U. M. Ünligil et al. (EMBO J. 19:5269-5280, 2000) reported crystallization of a GT13
representative. Interestingly, both have domain A, and the catalytic
residues identified were reported to be superimposable on D191 of SpsA
and therefore also E180 of WbbE.
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Abstract
Introduction
