 |
INTRODUCTION |
Surface carbohydrate structures are important virulence factors of
Gram-positive and Gram-negative bacteria. The Gram-negative opportunistic pathogen Pseudomonas aeruginosa causes fatal
lung infections in patients suffering from the inherited disorder
cystic fibrosis; it also affects burn victims and immunocompromised
individuals undergoing anticancer chemotherapy and those infected with
human immunodeficiency virus. This bacterium produces a variety of
polysaccharide structures, one of which is lipopolysaccharide
(LPS).1 LPS is composed of
three distinct units: (i) lipid A, which noncovalently binds the
molecule into the outer membrane, (ii) the core region that links, and
(iii) the O antigen, composed of linear repeats of di- to
hexasaccharide units, to the lipid A anchor (1). In the case of
P. aeruginosa, two different O antigens are made, namely
A-band and B-band (2). A-band is a homopolymer of
D-rhamnose present in most serotypes, whereas B-band is a
serospecific heteropolymer consisting of di- to pentasaccharide
repeating units. Differences in B-band repeating units are responsible
for the classification into 20 International Antigenic Typing System
serotypes (3, 4). Mutants devoid of O antigen are 1000-fold less
virulent than wild-type bacteria (5).
Staphylococcus aureus is an important bacterial pathogen
responsible for a broad spectrum of human and animal diseases including cutaneous as well as wound infections and more life-threatening infections such as endocarditis and bacteremia. Moreover, S. aureus produces numerous exotoxins, some of which cause diseases
such as toxic shock syndrome and food poisoning (6). The majority of
clinical S. aureus isolates produce either a type 5 or type 8 capsule (CP), which renders the organisms resistant to phagocytic uptake (7). Moreover, CP production has been shown to enhance virulence
in animal models of infection (8). S. aureus is highly efficient at acquiring resistance to antibiotics; the first documented case of a vancomycin-resistant S. aureus infection in a
United States patient was recently reported by the Centers for Disease Control (60).
L-FucNAc has thus far been described exclusively as a
constituent of bacterial polysaccharide structures. It is part of the O
antigen of P. aeruginosa serotypes O4, O11, and O12, of the CP of S. aureus serotypes 5 and 8, and of
Streptococcus pneumoniae CP type 4 (9-12). LPSs of
Escherichia coli O26 and CP of Bacteroides fragilis also contain L-FucNAc (13, 14). It should be
noted that not all L-FucNAc-containing bacteria are listed
above; for the scope of this study, only those bacteria for which
sequence data of the corresponding polysaccharide biosynthesis gene
clusters are available have been taken into consideration (E. coli O26 polysaccharide cluster data are referred to in Ref. 15).
With respect to P. aeruginosa, the
L-FucNAc-containing strains, particularly International
Antigenic Typing System O4 and O11, belong to the most clinically
prevalent strains besides serotypes O3 and O6 (16). The O11 strain
PA103 is a high level exotoxin A producer (17). S. aureus CP
serotypes 5 and 8 make up ~80% of clinical isolates (18), and
L-FucNAc is a component of both CP structures. Moreover,
both P. aeruginosa and S. aureus are associated
with nosocomial infections, as well as being known for resistance
against antibiotics. S. pneumoniae type 4 capsule is a
component of a heptavalent streptococcal vaccine, which is available
commercially (19). These data implicate the importance of the
L-FucNAc residue in pathogenicity. Targeting its
biosynthesis could lead to the development of therapeutic agents that
affect important virulence factors of Gram-positive and Gram-negative bacteria.
To date, the biosynthesis pathway leading to the nucleotide-activated
precursor of L-FucNAc is not clearly defined. Two putative pathways have been proposed, both lacking any experimental evidence. Lee and Lee (20) suggested a three-step route starting from UDP-D-ManNAc and involving (i) 3-epimerization, (ii)
5-epimerization, and (iii) 6-dehydration leading to
UDP-L-FucNAc. Jiang et al. (15) proposed a
reaction scheme analogous to the biosynthesis of
GDP-L-fucose from GDP-D-mannose. This group
suggested that UDP-D-GlcNAc is first converted to
UDP-D-ManNAc to get the same precursor as in the above
pathway. The next reaction step is 4,6-dehydration to get
UDP-2-acetamido-2,6-dideoxy-D-lyxo-4-hexulose,
and finally a bifunctional enzyme would catalyze 3,5-epimerization and
4-reduction to yield UDP-L-FucNAc. The S. aureus
genes cap5/8E, cap5/8F, and cap5/8G or the homologous S. pneumoniae genes cps4M(J), cps4N(K), and
cps4L have been proposed to be involved in this biosynthesis (15, 20).
We present for the first time a proposed biosynthetic pathway for
nucleotide-activated L-FucNAc, namely
UDP-L-FucNAc from its precursor UDP-D-GlcNAc.
We provide evidence that three P. aeruginosa enzymes, WbjB,
WbjC, and WbjD (and their S. aureus homologs
Cap5E, Cap5F, and Cap5G) catalyze a five-step reaction cascade.
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EXPERIMENTAL PROCEDURES |
Materials--
UDP-N-acetyl-D-glucosamine,
UDP-N-acetyl-D-galactosamine,
UDP-D-glucose, NAD+, NADH, NADP+,
NADPH, and the antibiotics used in this study were obtained from
Sigma-Aldrich. HiTrap Chelating columns were purchased from Amersham
Biosciences, Econo-Pac High Q columns were from Bio-Rad, and pET28a,
pET24a+, and pET24c+ were from Novagen (Madison, WI).
Bacterial Strains, Cloning Vectors, and Growth
Conditions--
The bacterial strains and plasmids used in this study
are listed in Table I. P. aeruginosa strains were grown in Luria broth (Invitrogen) or on
Pseudomonas Isolation Agar (Difco Laboratories, Detroit,
MI). S. aureus was grown in tryptic soy broth (Difco Laboratories). E. coli strains Top10 (Invitrogen) and JM109
(21) were used for plasmid propagation. E. coli strain SM10
(22) was used as donor in bacterial conjugation. For protein
overexpression E. coli strain BL21(DE3) was used (Novagen).
Overexpression was induced by the addition of
isopropyl-1-thio-
-D-galactopyranoside (Invitrogen) at a
final concentration of 1 mM. The media were supplemented
with ampicillin (100-250 µg/ml), kanamycin (25 µg/ml or 50 µg/ml), carbenicillin (250 µg/ml for E. coli and 300 µg/ml for P. aeruginosa), gentamicin (15 µg/ml for
E. coli and 300 µg/ml for P. aeruginosa),
tetracycline (15 µg/ml for E. coli and 100 µg/ml for
P. aeruginosa), chloramphenicol (10 µg/ml), or
erythromycin (10 µg/ml) when necessary.
Analytical Techniques--
SDS-PAGE was done according to
the method of Laemmli (23) with slight modifications. Gels were stained
with Coomassie Brilliant Blue R-250 (Sigma-Aldrich). The protein
concentrations were determined as described by Bradford (24). LPS was
prepared according to the method of Hitchcock and Brown (25). The
samples were run on a 12% SDS-PAGE gel, and the LPS was either
transferred to a nitrocellulose membrane or visualized by a rapid
silver staining procedure (26). Antibodies MF55-1 (mAb to B-band O
antigen of serotype O11) (27), grouping antiserum O11 (polyclonal
antibody to B-band O antigen of serotype O11; Chengdu Institute of
Biological Products, Chengdu, China), and N1F10 (mAb to A-band O
antigen) (28) were used for Western immunoblotting analysis. The blots were developed with anti-mouse immunoglobulin G alkaline phosphatase (Jackson Immunoresearch, West Grove, PA) for mAbs, and anti-rabbit immunoglobulin G alkaline phosphatase (Bio-Rad) for the polyclonal antiserum. 5-Bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium (Sigma-Aldrich) were used as the alkaline phosphatase substrate.
Colony immunoblot analysis was performed as previously described (29)
with the use of CP5-specific polyclonal rabbit antiserum. The blots
were developed using protein A-horseradish peroxidase conjugate
(Zymed Laboratories Inc., South San Francisco, CA) and the Bio-Rad horseradish peroxidase conjugate substrate kit.
Sequence Analysis--
BLAST (30) and Multalin (31) were used
for analysis of nucleotide and protein sequences.
DNA Manipulations, PCR, DNA Sequencing, and Protein
Sequencing--
All of the standard DNA recombinant procedures
were performed according to the methods described by Sambrook et
al. (32) or as recommended by the corresponding manufacturer. PCR
was carried out with a GeneAmp PCR System 2400 (PerkinElmer Life
Sciences). DNA sequencing was performed by Mobix Lab (Hamilton, Canada)
or by the Beth Israel Deaconess Molecular Medicine Unit (Boston, MA).
N-terminal protein sequencing was performed by the Dana Farber Molecular Biology Core Facility (Boston, MA).
Plasmid Construction--
The oligonucleotide primer sequences
are given in Table II. Chromosomal DNA of
P. aeruginosa International Antigenic Typing System strain
O11 or S. aureus capsular type 5 was used for amplification of the genes. Pwo polymerase (Roche Molecular Biochemicals) was used
for PCR amplification. Primer pairs wbjB1F/wbjB2R, wbjC1F/wbjC2R, wbjD1F/wbjD2R, and capE1F/capE2R were used for amplification of the
wbjB, wbjC, wbjD, and cap5E
(clinical isolate provided by A. Chow, Vancouver, Canada) genes,
respectively. The PCR products of genes wbjD and
cap5E were digested with NcoI and
BamHI and ligated into the modified pET23 derivative (33),
cut with the same enzymes. PCR product wbjC was cut with
NdeI and HindIII and ligated into appropriately
digested pET28a. Finally, PCR product wbjB was digested with
NdeI and EcoRI and ligated into
NdeI/EcoRI-cut pET28a. The corresponding plasmids
pFuc11 (wbjB), pFuc12 (wbjC), pFuc13
(wbjD), and pFuc21 (cap5E) were confirmed by DNA
sequencing and used for amplification of PCR products for
complementation studies and/or overexpression of N-terminal
histidine-tagged fusion proteins.
S. aureus cap5E, capF, and capG genes
were PCR-amplified from strain Newman chromosomal DNA with High
Fidelity PCR Supermix (Invitrogen). Primers 5E-F and 5E-R were utilized
to amplify the cap5E gene. This amplicon was digested with
XhoI and NheI and ligated to pET24a+ digested
with the same enzymes. The resultant plasmid pKBK50d contained the
ribosomal-binding site and the ATG start site of the pET vector
in-frame with the entire cap5E gene and C-terminal
six-histidine tag. A similar strategy was applied to clone the
cap5F gene; it was amplified with primers 5F-F and 5F-R and
cloned into the pET24a+ vector, creating plasmid pET5F1.1. The
cap5G gene was amplified with KK1 and KK2. The 1.18-kb
amplicon was digested with XbaI and EcoRI and
ligated to appropriately digested pET24c+, yielding plasmid pKBK6a with
a C-terminal histidine tag. All of the plasmids were confirmed by
nucleotide sequencing of the PCR-amplified genes.
To construct the plasmids containing a nonpolar gene disruption of
P. aeruginosa wbjB, the PCR product described above was ligated into SmaI-cut pEX100T, taking advantage of the
blunt-end PCR product made by Pwo polymerase. The
wbjB-containing plasmid pKOB1 was subsequently cut with
KpnI, and the aacC1 gene (gentamicin resistance
cassette) from pUCGm was ligated into pKOB1 to yield the
wbjB knockout plasmid pKOB2.
To create a plasmid for S. aureus cap5G gene disruption,
subclone pJCL69 (containing the end of cap5E through the
start of cap5I) was digested with BclI, releasing
a 294-bp fragment 50 bp downstream of the ATG start site of
cap5G yielding pJCL69-2. The ermB gene from pERMB
was ligated into the BclI site of pJCL69-2 to create
pJCL69-2ermB. A 4402-bp XbaI-BamHI fragment from
pJCL69-2ermB was ligated to digested pCL10 to yield pKOR1. To create a
knockout plasmid for use in cap5F allelic replacement, a
modified pCL10 plasmid containing ermB in the
BamHI site was constructed (pAP1.2EX). A 1.6-kb region of
the cap5 gene locus (containing 450 bp of cap5F gene and 1124 bp of the cap5G gene) was PCR-amplified using
primers 12402F2 and 12402R2, digested with XbaI, and ligated
to XbaI-digested pAP1.2EX to form pKOR3. A 1941-bp region of
the cap5 locus containing 385 bp of cap5D, the
complete open reading frame (1026 bp) of cap5E gene, and 530 bp of cap5F was PCR-amplified using primers 12402F1 and
12402R1. This amplicon was digested with SacI and EcoRV and ligated to similarly digested pKOR3 to yield the
11.32-kb plasmid pKOR4.
pFuc11 and pFuc21 plasmid DNA was used to amplify the genes for the
complementation plasmids. PCR was carried out with T7 forward primer
and the corresponding reverse primers used to get the expression
plasmids. The XbaI site in the T7 promoter sequence was used
to ligate the PCR product into pUCP27 digested with
XbaI/EcoRI for wbjB or
XbaI/KpnI in the case of cap5E. Thus,
we could take advantage of the ribosomal-binding site of the pET
vectors and achieve complementation with the N-terminally
histidine-tagged fusion proteins. The resulting plasmids pPAB27 and
pSAE27 were confirmed by DNA sequencing. S. aureus
plasmid pKOR2 was constructed by ligating a 2.2-kb
BamHI-XbaI fragment from pJCL43 containing the
cap5G gene along with 920 bp of upstream and 144 bp of
downstream flanking sequences into the E. coli/S.
aureus shuttle vector pLI50.
Protein Overexpression and Purification--
Expressions were
carried out at 37 °C using terrific broth (32), supplemented with 50 µg/ml kanamycin for WbjB and WbjC, with 25 µg/ml kanamycin for
Cap5E, Cap5F, or Cap5G, or with 250 µg/ml ampicillin for WbjD. The
cultures were grown to an A600 nm of 0.6, and
expression was induced with
isopropyl-1-thio-
-D-galactopyranoside at a final
concentration of 1 mM. WbjB ,WbjD, Cap5E, and Cap5F expression was carried out for 3 h at 37 °C, WbjC was expressed for 4 h at 37 °C, and Cap5G was expressed for 4 h at
30 °C. The cells were disrupted on ice by ultrasonication. Cell
debris and membrane fractions were removed by ultracentrifugation at
300,000 × g. Purifications using nickel chelating
columns were performed as recommended by the manufacturers. Purified
WbjC and WbjD could be obtained at 200 mM imidazole,
whereas WbjB, Cap5E, Cap5F, and Cap5G were eluted from the column at
300 mM imidazole. Dithiothreitol was added to a final
concentration of 1 mM, and the proteins were stored at
20 °C after the addition of 40% glycerol. The purity of the
enzymes was checked by SDS-PAGE analysis on a 10% gel, and N-terminal
protein sequencing was performed on Cap5E, Cap5F, and Cap5G to verify
the purified proteins.
Capillary Electrophoresis (CE) Analysis of
UDP-L-FucNAc Biosynthesis--
CE analysis was performed
with a P/ACE MDQ Glycoprotein system with UV detection (Beckman
Coulter, Fullerton, CA). The samples were separated at 22 kV in 25 mM borate buffer (pH 9.5) at 25 °C. Typically, the
reactions contained 0.5 mM UDP-D-GlcNAc, 0.1 mM NADP+, and an excess of hydride donor (NADH
or NADPH). For CE-MS analysis, the UDP-D-GlcNAc
concentration was 0.1 or 0.5 mM. The standard reaction
buffer was 20 mM Tris-HCl at pH 8.0 supplemented with 10 mM MgCl2. The enzymes (~1 µg each) were
added, and the reaction mixtures were incubated at 37 °C for 60 min
before being analyzed by CE. The reaction cascades were investigated as
stepwise reactions adding one enzyme at a time (allowing optimal
substrate conversion on each reaction step) and as combined reactions
adding more than one enzyme to start the reaction.
Coupled CE-MS Analysis of UDP-L-FucNAc
Biosynthesis--
The crystal model 310 CE instrument (ATI Unicam,
Boston, MA) was coupled to an API 3000 mass spectrometer (MDS/Sciex,
Concord, Canada) via a micro Ionspray interface. A sheath solution
(isopropanol:methanol, 2:1) was delivered at a flow rate of 1 nl/min to
a low dead volume tee (internal diameter, 250 µm; Chromatographic
Specialties, Brockville, Canada).
The separation was achieved using a 90-cm bare fused silica capillary
with a buffer of 30 mM morpholine/formic acid in deionized water, pH 9.0, with 5% methanol. For positive detection mode, a 10 mM ammonium acetate buffer, pH 9.0, containing 5% methanol was used. A separation voltage of 30 kV was typically applied at the
injection end of the capillary. The outlet of the capillary was tapered
to a ~15-µm diameter with a laser puller (Sutter Instruments, Novato, CA).
Mass spectra were acquired using a dwell time of 3.0 ms/step of 1 m/z unit in full mass scan mode. For CE-MS/MS
experiments, a sample loading of ~25 nl was applied. The MS/MS data
were acquired with dwell times of 3.0 ms/step of 1 m/z unit on the triple quadrupole instrument.
Collisional activation of selected precursor ions in the
radiofrequency-only quadrupole collision cell was achieved using
nitrogen as a target gas. Further information was obtained by
conducting MS/MS experiments on the second generation fragment ions (or
MS/MS/MS), promoted by front end collision-induced dissociation. These
selected ions were formed by increasing the orifice voltage (from 60 to
180 V, in this case).
Synthesis and Preparation of Intermediate Products--
5.5
µmol (3.5 mg) of UDP-D-GlcNAc were converted
quantitatively to a mixture of keto-sugars using 100 µg of WbjB. The
reaction was carried out in 20 mM triethylammonium
bicarbonate buffer (pH 8.0) for 4.5 h at 37 °C. The protein was
removed by ultrafiltration with Centriplus cartridges (Millipore,
Bedford, MA). For analysis of the keto-mixture, the sample was
acidified to pH 4.5 with Dowex 50 (Bio-Rad) to get rid of
CO2 and concentrated to 100 µl by lyophilization. This
concentrated compound was analyzed by NMR. The keto-sugar that
migrated faster than NADP+, as judged by CE analysis, was
purified over an Econo-Pac High Q anion exchange column with a linear
triethylammonium bicarbonate gradient (0-500 mM).
Fractions containing the keto-sugar were pooled, acidified with Dowex
50, lyophilized completely, and analyzed by NMR.
5.5 µmol (3.5 mg) of UDP-D-GlcNAc were converted
quantitatively to the putative
UDP-2-acetamido-2,6-dideoxy-L-talose using 200 µg each of
WbjB and WbjC and equimolar amounts of NADPH. The reaction was
performed overnight in 20 mM Tris buffer (pH 8.0) at
37 °C. The enzyme from the reaction mix was removed by
ultrafiltration with Centriplus cartridges. The purification of the
reaction product was performed as described above for the
keto-intermediate. The fractions corresponding to
UDP-2-acetamido-2,6-dideoxy-L-talose were pooled, acidified
to pH 4.5 with Dowex 50, concentrated to a final volume of 80 µl by
lyophilization, and used for NMR analysis. The yield was ~3 mg of
UDP-2-acetamido-2,6-dideoxy-L-talose (85%).
NMR Analysis of Intermediate Products--
All of the spectra
were acquired using a Varian Inova 500 MHz spectrometer equipped with a
Z-gradient 3-mm triple resonance (1H, 13C,
31P) probe. The lyophilized UDP-xylo-sugar
sample was dissolved in 140 µl of 99% D2O. The other
samples, dissolved in 80-100 µl of H2O, were prepared by
adding D2O up to a volume of 140 µl. The experiments were
performed at 25 °C with suppression of the deuterated
H2O signal at 4.78 ppm. The methyl resonance of
acetone was used as an internal or external reference at 2.225 ppm for 1H spectra and 31.07 ppm for 13C spectra.
Standard homo- and heteronuclear correlated two-dimensional pulse
sequences from Varian, COSY, HSQC, HMBC, and 31P HMQC were
used for general assignments (34). Selective one-dimensional TOCSY with
a Z-filter and one-dimensional NOESY experiments were performed for
complete residue assignment and for the determination of
1H-1H nuclear Overhauser enhancements (35). A
mixing time of 80 ms was used for the one-dimensional TOCSY experiments
and 800 ms for the one-dimensional NOESY experiments.
Nonpolar Gene Knockout of wbjB--
The
wbjB::aacC1-containing plasmid pKOB2
was transformed into the mobilizing E. coli strain SM10, and
the construction of the wbjB knockout mutant was
accomplished as was described by Schweizer and Hoang (36). Gene
replacement was confirmed by resistance to gentamicin, resistance to
sucrose, susceptibility to carbenicillin, and PCR. Primer pairs
wbjB1F/wbjB2R, wbjB1F/wbjC2R, wbjB1F/gent2R, gent1F/wbjB2R, and
wzy11F/wbjB2R (Table II) were used to prove nonpolar insertion of the
aacC1 gene into the B-band O antigen locus. wzy11F is a
forward primer homologous to sequence located directly upstream of
wbjB, and gent1F and gent2R are forward and reverse primers
within the gentamicin resistance gene aacC1. Western
immunoblotting analysis provided further evidence of the successful
mutation of the gene.
Complementation of the wbjB Gene Knockout--
The plasmids
pPAB27 and pSAE27, which have the wbjB or cap5E
in the opposite direction as the lac promoter (resulting in
lower expression levels of the cloned genes), were transformed into the
knockout strain BK103B by electroporation. The cultures were grown
overnight, and LPS was analyzed by methods described above.
Nonpolar Gene Knockout of capF and capG--
Plasmids pKOR1 and
pKOR4 were separately electroporated into S. aureus RN4220
(37) and transduced with phage 80
(38) into S. aureus
Newman, in both cases selecting for Cmr colonies at
30 °C. Allelic replacements were performed as described by Kiser
et al. (39). Southern blot analysis confirmed the
identity of cap5G mutant G01 and cap5F mutant F4.
Colony immunoblot analysis of CP5 expression was performed as
previously described (29).
Complementation of the cap5G and cap5F Gene
Knockouts--
Plasmid pKOR2 carrying cap5G was transformed
into S. aureus RN4220 and then transduced into S. aureus Newman G01. The S. aureus vector pLI50 was
similarly introduced into strain G01 as a control. The cap5F
knockout mutant (strain F4) was complemented in trans with
pCAP17, a plasmid containing the cap5 promoter region and intact cap5A through cap5F in pCUI. pCAP16, a
similar pCU1 construct containing the cap5 promoter and
cap5A through cap5E, was utilized as a negative control.
 |
RESULTS |
Sequence Analyses--
A common feature of bacterial gene clusters
associated with the biosynthesis of L-FucNAc is the
presence of three consecutive genes with no assigned function (Table
III). These genes are also part of the
genomes of Pasteurella multocida PM70 and Leptospira interrogans serovars Copenhageni and Pomona. Whether these two bacteria contain L-FucNAc in their surface polysaccharide
is not known. The proteins encoded by wbjB/cap5E
and wbjC/cap5F or their homologs belong to the
SDR protein family (40). Both WbjB/Cap5E and WbjC/Cap5F have the
nucleotide-binding signature GXXGXXG near their N
terminus (referred to as the Rossmann fold) essential for binding of
the cofactor NAD+ or NADP+ (41). Another
typical feature of the SDR protein family is the so-called catalytic
triad, SMK in the case of WbjB/Cap5E and SYK in WbjC/Cap5F. WbjB/Cap5E
is moderately homologous to WbpM, which is putatively involved in
UDP-N-acetyl-D-fucosamine/quinovosamine biosynthesis and shows reactivity on UDP-D-GlcNAc (42).
Thus, we postulated WbjB/Cap5E to be the enzyme catalyzing the first reaction step involving UDP-D-GlcNAc. Because proteins of
this family are highly homologous, is very difficult to predict their exact function. The third putative L-FucNAc biosynthesis
protein is WbjD/Cap5G, which shows homology to UDP-GlcNAc 2-epimerases involved in the biosynthesis of UDP-D-ManNAc (39). To
determine whether the gene products of
wbjB/cap5E, wbjC/cap5F, and
wbjD/cap5G are actually involved in the
biosynthesis of nucleotide-activated L-FucNAc and to
determine the order in which they would act in the pathway, two
approaches have been used: overexpression of the corresponding proteins
to carry out in vitro enzyme assays and knocking out the
genes, evaluating the phenotype of the mutants, and performing
complementation studies.
Expression and Purification of the UDP-L-FucNAc
Biosynthesis Enzymes--
WbjB, WbjC, WbjD, Cap5E, and Cap5F were
expressed at high levels at 37 °C. High level expression resulting
in soluble Cap5G could only be achieved at 30 °C. After sonication
and ultracentrifugation, the majority of the proteins proved to be in
the supernatant, enabling high yield affinity purification on chelating
columns. After addition of 40% glycerol, the enzymes were stable at
20 °C for several weeks. The apparent molecular masses
(Mr) of the proteins, determined by SDS-PAGE,
were in good agreement with the calculated molecular masses,
i.e. WbjB, 40.7 kDa; WbjC, 43.6 kDa; and WbjD, 43.9 kDa
(Fig. 1A). Purified
recombinant Cap5E, Cap5F, and Cap5G are shown in Fig. 1B.
The predicted molecular masses of these recombinant proteins are 39.9, 43.7, and 45.1 kDa, respectively. N-terminal protein sequencing of
Cap5E (MFDDKILL), Cap5F (LTLNIVIT), and Cap5G (MEKLKLMTIV) correlated
with the predicted initial eight to ten amino acids for these proteins.
The purified proteins were used for functional characterization of the
biosynthesis pathway of nucleotide-activated L-FucNAc.

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Fig. 1.
SDS-PAGE analysis of purified
UDP-L-FucNAc biosynthesis enzymes. A,
P. aeruginosa International Antigenic Typing System O11.
B, S. aureus serotype 5. His6 refers
to the hexahistidine tag at the N or C terminus. Prestained protein
molecular weight standards (Invitrogen) or the Low Range SDS-PAGE
Molecular Weight Standard (Bio-Rad) were used.
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WbjB/Cap5E Is a Trifunctional Enzyme Catalyzing the
First Three Enzymatic Reactions--
Sequence analysis suggested that
WbjB was the first enzyme in the pathway with UDP-D-GlcNAc
as substrate. Indeed, in the WbjB enzyme-substrate reaction, the peak
corresponding to UDP-D-GlcNAc disappeared almost
completely, and two new peaks appeared on CE analysis (Fig.
2B). NADP+ was an
essential cofactor in WbjB catalysis. Addition of NAD+ or
the absence of any added cofactor resulted in less than 5% substrate
conversion. Despite performing time course experiments to determine
which of the two peaks appeared first, it was not possible to make any
assignment. Both products were formed simultaneously in a 1:3 ratio
(Fig. 2B, intermediates 1 and 2). The
achieving of an equilibrium would be characteristic of an epimerase
reaction. Two other related sugar nucleotides,
UDP-N-acetyl-D-galactosamine and
UDP-D-glucose, also were tested as substrates with WbjB and cofactor, but no reaction could be detected. To further verify that
WbjB is the enzyme catalyzing the first reaction step, purified WbjC or
WbjD were also incubated with UDP-D-GlcNAc; again no
substrate conversion was discerned.

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Fig. 2.
CE analysis of UDP-L-FucNAc
biosynthesis. A, standard, UDP-D-GlcNAc,
NADP+, and NADPH. B, UDP-D-GlcNAc,
converted with WbjB and NADP+ (intermediates 1 and 2 represent the two 2- amino-4-keto-2,6-dideoxy-hexose
intermediates). C, UDP-D-GlcNAc, converted with
WbjB, WbjC, NADP+, and NADPH (UDP-TNAc refers to
UDP-2-acetamido-2,6-dideoxy-L-talose). D,
UDP-D-GlcNAc, converted with WbjB, WbjC, WbjD,
NADP+, and NADPH. E, UDP-D-GlcNAc,
converted with WbjB, WbjC, NADP+, and NADH
(NAD+ and NADH have not been added to the standard
A; the reason being that NADH comigrates with
NADP+, and NAD+ results in two peaks).
F, UDP-D-GlcNAc, converted with WbjB, WbjC,
WbjD, NADP+, and NADH. a.u., arbitrary
units.
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CE-MS was used to characterize further the two reaction products formed
by WbjB. Although the reaction appeared to be close to quantitative,
only a minor part of UDP-D-GlcNAc was converted to a
product with a molecular mass of 588 (negative mode; Fig. 3B), the expected mass if the
first reaction step involved 4,6-dehydration. Table
IV lists parent and fragment ions
obtained in CE-MS analyses. However, CE-MS/MS in the positive mode at
m/z 608 clearly demonstrated that the product
formed in the WbjB catalyzed reaction with the same molecular mass as
UDP-D-GlcNAc was not UDP-D-GlcNAc (Fig. 4, A and B). The
fragment ion at m/z 138 characteristic of
N-acetyl-hexose sugars having a hydroxyl group at C-6
disappeared almost completely. A time course experiment was performed
to investigate this reaction further. CE-MS/MS/MS was carried out at
m/z 608 (first generation ions) through
m/z 204 (second generation ions) to analyze the hexose moiety of the nucleotide-activated sugar. Indeed, a significant decrease of the fragmentation ion at m/z 138 was
observed (Fig. 5). The presence of a
6-deoxy-sugar with a molecular mass equivalent to that of its parent
6-hydroxy-sugar provided the evidence for the addition of water
somewhere on the hexose moiety. CE-MS/MS analysis of the peak at
m/z 590 (positive mode) demonstrated that the
second reaction product is UDP-2-acetamido-4-keto-2,6-dideoxy sugar in
the nonhydrated form (Fig. 4C). Both CE and CE-MS analyses also were performed using the S. aureus homolog Cap5E, with
results identical to those obtained for WbjB (data not shown).

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Fig. 3.
CE-MS analysis of UDP-L-FucNAc
biosynthesis in the negative mode. A,
UDP-D-GlcNAc. B, UDP-D-GlcNAc,
converted with WbjB and NADP+. C,
UDP-D-GlcNAc, converted with WbjB, WbjC, NADP+,
and NADPH. D, UDP-D-GlcNAc, converted with WbjB,
WbjC, WbjD, NADP+, and NADPH. Peaks at
m/z 563, 545, and 547 are due to the UDP moiety.
The peak at m/z 742 results from
NADP+, and the peaks at m/z 610, 612, and 628 are due to mono-sodium salts of m/z 588, 590, and 606, respectively.
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Fig. 4.
CE-MS/MS analysis of UDP-L-FucNAc
biosynthesis in the positive mode. A,
UDP-D-GlcNAc. B and C,
UDP-D-GlcNAc, converted with WbjB and NADP+.
D, UDP-D-GlcNAc, converted with WbjB, WbjC,
NADP+, and NADPH (UDP-D-GlcNAc, converted with
WbjB, WbjC, WbjD, NADP+, and NADPH looks exactly the same
as D).
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Fig. 5.
Time course investigation of the
WbjB-catalyzed reaction using CE-MS/MS/MS analysis in the positive
mode. UDP-D-GlcNAc was reacted with WbjB and
NADP+. MS/MS/MS at m/z 608 (first
generation ions) through m/z 204 (second
generation ions) was recorded at the time points indicated.
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To obtain unequivocal evidence of the identity of these intermediates,
NMR experiments have been performed. The putative keto-intermediate that migrated faster than NADP+ was purified by anion
exchange chromatography. It was lyophilized to dryness and dissolved in
D2O. Although it was fairly unstable, we were able to
assign the structure of this intermediate sugar nucleotide; the
1H spectrum is presented in Figs.
6A and
7A. One-dimensional selective experiments on the sugar resonances yielded accurate coupling constants. A value of J1,2 of 3.6 Hz indicated
that H-1 was equatorial, having the
-configuration. A large
J2,3 value of 10.8 Hz indicated that H-2 and H-3
were trans to each other. The strong NOE between H-5 and H-3
indicated that H-3 and H-5 were both axial (Fig. 7D). From
the HSQC spectrum, the protonated carbons were assigned. From the HMBC
spectrum, the quaternary carbon at C-4 and the NAc-C=O resonances were
assigned. Because this sample was dissolved in pure D2O,
the NH resonance was not observed. The chemical shifts of hydrogens and
carbons, as well as the 1H-1H coupling
constants, are given in Table V. The
1H, 13C, and 31P chemical shifts
for the UDP moiety are similar to the one reported before (34). The
sugar nucleotide finally arrived at is
UDP-2-acetamido-2,6-dideoxy-
-D-xylo-4-hexulose.

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Fig. 6.
1H spectra of intermediates in
UDP-L-FucNAc biosynthesis. A, sample
containing
UDP-2-acetamido-2,6-dideoxy- -D-xylo-4-hexulose.
B, sample containing degradation products from a mixture of
UDP-2-acetamido-2,6-dideoxy- -D-xylo-4-hexulose
and
UDP-2-acetamido-2,6-dideoxy- -L-arabino-4-hexulose.
C, sample containing
UDP-2-acetamido-2,6-dideoxy- -L-talose. Varying amounts
of UMP were also present in each sample. Peaks from the
triethylammonium salt (1.3 and 3.2 ppm) and glycerol (3.6, 3.7, and 4.1 ppm) could also observed.
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Fig. 7.
NMR analysis of
UDP-2-acetamido-2,6-dideoxy- -D-xylo-4-hexulose.
A, 1H spectrum. B, one-dimensional
TOCSY (H-3). C, one-dimensional TOCSY (H-6). D,
one-dimensional TOCSY-NOESY (H-6 and H-5).
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The second intermediate detected by CE analysis could not be purified
in the same manner as the first because of instability. To analyze this
sugar, the WbjB reaction mix was concentrated on the lyophilizer after
removing the enzyme by ultrafiltration. D2O was added to
the concentrate, and the keto-mixture was analyzed by NMR (Figs.
6B and 8A).
Although no intact UDP-sugar could be detected because of degradation,
the structure of the putative keto-sugar could be resolved by analyzing
the degradation products. From a series of one-dimensional TOCSY
experiments on all of the anomeric peaks identified by a HSQC spectra,
a resonance at 5.24 ppm could be identified as belonging to an expected
keto-sugar. A small J2,3 value of 3.5 Hz
indicated that H-2 and H-3 are gauche to each other. The strong NOE
between H-1 and H-5 indicated that H-1 and H-5 were both axial (Fig.
8C). The lack of NOE between H-3 and H-5 and between H-3 and
H-1 indicated that H-3 is equatorial (Fig. 8, C and
D). From the HSQC spectrum the protonated carbons were
assigned. From the HMBC spectrum, the quaternary carbon at C-4 and the
NAc-C=O resonances were assigned. The NMR data are given in Table V.
The presence of this degradation product in this mixture is consistent
with the second intermediate being UDP-2-acetamido-2,6-dideoxy-
-L-arabino-4-hexulose.
The expected UDP-2-acetamido-2,6-dideoxy-
-L-lyxo-4-hexulose
could not be detected, either because of instability or because of an
equilibrium highly in favor of the two compounds described above.

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Fig. 8.
NMR analysis of
UDP-2-acetamido-2,6-dideoxy- -L-arabino-4-hexulose.
A, 1H spectrum of a sample containing
degradation products from a mixture of
UDP-2-acetamido-2,6-dideoxy- -D-xylo-4-hexulose
and
UDP-2-acetamido-2,6-dideoxy- -L-arabino-4-hexulose.
B, one-dimensional TOCSY (NH). C, one-dimensional
NOESY (H-1). D, one-dimensional NOESY (H-3).
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WbjC/Cap5F Reduces One of the 4-Keto-intermediates to
Yield a Nucleotide-activated 2-Acetamido-2,6-dideoxy Sugar--
When
UDP-D-GlcNAc was incubated with WbjB and WbjC in the
presence of NADP+ and NADPH, the two peaks produced in the
WbjB reaction decreased, and a new peak appeared in the chromatogram
comigrating with the precursor sugar UDP-D-GlcNAc (Fig.
2C). These reaction steps could also be performed
consecutively, first allowing conversion to the two 4-keto-intermediate
and subsequently adding WbjC and NADPH (Fig.
9C). NADPH and NADH could be
used equally well as hydride donors in the reduction reaction (Fig. 2,
C and E).

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Fig. 9.
CE analysis of UDP-L-FucNAc
biosynthesis. A, standard, UDP-D-GlcNAc,
NADP+ and NADPH. B, UDP-D-GlcNAc,
converted with WbjB and NADP+ (intermediates 1 and 2 represent the two 2,6-dideoxy-2-amino-4-keto-hexose
intermediates). C, UDP-D-GlcNAc, converted
with WbjB and NADP+, followed by WbjC and NADPH (UDP-TNAc
refers to UDP-2-acetamido-2,6-dideoxy-L-talose).
D, UDP-D-GlcNAc, converted with WbjB and
NADP+, followed by WbjC and NADPH, and followed by WbjD.
a.u., arbitrary units.
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The reduction reaction was further investigated by CE-MS analysis,
which in the negative mode showed a decrease of
m/z 606 (hydrated
2-acetamido-4-keto-2,6-dideoxy-sugar), the disappearance of
m/z 588 (2-acetamido-4-keto-2,6-dideoxy-sugar),
and the appearance of a new ion at m/z 590 (Fig.
3C). This is consistent with a reduction reaction.
Additional evidence was obtained by CE-MS/MS at
m/z 592 in the positive mode, resulting in the
expected fragmentation pattern (Fig. 4D). Again, CE as well
as CE-MS experiments using the Staphylococcus protein Cap5F
gave identical results (data not shown).
Finally, the reduction product was purified by anion exchange
chromatography and analyzed by NMR spectroscopy (Figs. 6C
and 10A). From
one-dimensional TOCSY experiments, the small coupling constants for
protons within the ring indicated a talose configuration. The observed
coupling constants for J1,2 = 1.5 Hz,
J2,3 = 3.2 Hz, J3,4 = 3.6 Hz, and J4,5
1 Hz were similar to their
respective J values of 1.5, 3.0, 3.0, and 1.0 Hz observed
for synthetic talose oligosaccharides (43). The strong NOE between H-1
and H-3, between H-1 and H-5, and between H-3 and H-5 indicated that
H-1, H-3, and H-5 were axial (Fig. 10, C and D).
From the HSQC spectrum, the protonated carbons were assigned. From the
HMBC spectrum, the NAc-C=O resonances were assigned. The NMR data are
given in Table V. Taken together, the reduction product generated by
WbjC is UDP-2-acetamido-2,6-dideoxy-
-L-talose.

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Fig. 10.
NMR analysis of
UDP-2-acetamido-2,6-dideoxy- -L-talose.
A, 1H spectrum. B, one-dimensional
TOCSY (NH). C, one-dimensional NOESY (H-1). D,
one-dimensional NOESY (H-3).
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WbjD/Cap5G Is a Putative C-2-Epimerase Catalyzing the
Formation of UDP-L-FucNAc--
As described above, WbjD
shows moderate homologies to UDP-D-GlcNAc 2-epimerases.
Indeed, this enzyme appears to catalyze an equilibrium reaction, as
would be expected of an epimerase. The peak corresponding to the
2-acetamido-2,6-dideoxy sugar synthesized in the first three reaction
steps decreased significantly, and a new peak migrating slightly faster
appeared (Fig. 2, D and F). Approximately 45% of
the reduction product was converted to this new peak. Again, this final
product could be observed either when all enzymes and cofactors were
added simultaneously at the beginning of the reaction or when the
reaction steps were carried out consecutively (Fig. 9D).
CE-MS analysis was carried out to investigate this new product. As
expected from an epimerization reaction, no changes could be observed
compared with the mass spectra obtained from the WbjC-catalyzed reaction (Fig. 3D). The fragmentation pattern in CE-MS/MS
analysis at m/z 592 (positive mode) looked
exactly the same as before the epimerization step (Fig. 4D;
m/z 592 spectra only shown once). This is
consistent with the formation of the 2-epimer of UDP-TalNAc, i.e. UDP-L-Fuc2NAc. The
Staphylococcus homolog Cap5G yielded the same results,
except for inactivity of the enzyme at 37 °C. The Cap5G reaction had
to be performed at 30 °C (data not shown).
Three Enzymes Catalyze a Five-step Reaction Cascade Converting
UDP-D-GlcNAc to a Product Consistent with
UDP--
L-FucNAc
We propose the following reaction scheme
for the biosynthesis pathway of nucleotide-activated
L-FucNAc (Fig. 11). WbjB/Cap5E catalyzes 4,6-dehydration, 5-epimerization, and
3-epimerization of the precursor UDP-D-GlcNAc, resulting in
as many as three intermediates, namely
UDP-2-acetamido-2,6-dideoxy-D-xylo-4-hexulose,
UDP-2-acetamido-2,6-dideoxy-L-arabino-4-hexulose, and
UDP-2-acetamido-2,6-dideoxy-L-lyxo-4-hexulose,
which are present in the ketal form. The next step is reduction at C-4
to yield UDP-2-acetamido-2,6-dideoxy-L-talose, catalyzed by
WbjC/Cap5F. Finally, the
UDP-2-acetamido-2,6-dideoxy-L-talose 2-epimerase WbjD/Cap5G
partially converted UDP-2-acetamido-2,6-dideoxy-L-talose to
a product that is consistent with UDP-L-FucNAc in a
C-2-epimerization reaction.

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Fig. 11.
Proposed biosynthesis pathways leading to
UDP-L-FucNAc. I,
UDP- -D-GlcNAc. II,
UDP-2-acetamido-2,6-dideoxy- -D-xylo-4-hexulose.
III,
UDP-2-acetamido-2,6-dideoxy- -L-arabino-4-hexulose,
IV,
UDP-2-acetamido-2,6-dideoxy- -L-lyxo-4-hexulose.
V, UDP-2-acetamido-2,6-dideoxy- -L-talose.
VI, UDP- -L-FucNAc.
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Inactivation of wbjB Abrogates B-band O Antigen Production in P. aeruginosa PA103--
A nonpolar gene knockout of wbjB has
been constructed by insertional mutation with a gentamicin resistance
cassette and allelic replacement. Strain BK103B with
wbjB::aacC1 on its chromosome no longer
produces B-band O antigen. In the silver-stained LPS-SDS-PAGE gel, only
the banding pattern of A-band O antigen is visible (Fig. 12A). The presence of A-band
O antigen was confirmed by Western immunoblotting analysis using the
mAb N1F10 (Fig. 12D). Moreover, LPS of the mutant does not
react with polyclonal or mAbs to O11 B-band (Fig. 12, B and
C). It has to be noted that mAb MF55-1 reacts with low
molecular weight O11 B-band. Production of the serospecific O antigen
in the mutant strain BK103B could be restored by adding P. aeruginosa wbjB or the S. aureus gene cap5E
on an E. coli/P. aeruginosa shuttle vector (Fig. 12,
A-C).

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Fig. 12.
Knockout and complementation analysis of
wbjB. A, silver-stained LPS-SDS-PAGE
gel. B, Western immunoblotting analysis with polyclonal
antiserum to B-band O antigen of serotype O11. C, Western
immunoblotting analysis with mAb MF55-1 (specific against B-band O
antigen of serotype O11). D, Western immunoblotting analysis
with mAb N1F10 (specific for A-band O antigen).
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Knockout of cap5E or cap5F and cap5G Results in Nonencapsulated S. aureus Mutants--
S. aureus strain NCTC 8325 carries a
complete cap5 locus but fails to produce CP5. Genomic
analysis of the cap5E gene from this strain (sequence of
8325 available at www.genome.ou.edu/staph.html) revealed that it
carried a single amino acid mutation (Met134 to
Arg134) within Cap5E. Wann and colleagues (44) demonstrated
that cap5E provided in trans restored CP5
expression to NCTC 8325-4. We insertionally inactivated the
cap5F gene by deleting 126 bp of the gene and replacing it
with an ermB cassette. Allelic replacement of the native
gene with this disrupted copy resulted in a CP-negative phenotype.
Complementation of this mutation was observed when a plasmid containing
the cap5 promoter region and genes cap5A through
cap5F was introduced into mutant F4. No complementation was
observed with the same plasmid containing only the cap genes 5A through 5E. Mutagenesis of cap5G in
strain Newman was achieved by deleting a 294-bp region of the gene and
replacing it with an ermB cassette. The resultant strain GO1
displayed a CP-negative phenotype that could be restored by
cap5G cloned in the vector pLI50. Fig.
13 shows a colony immunoblot of
cap5E, cap5F, and cap5G mutants that
failed to produce CP and the complemented strains with the restoration
of CP production.

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Fig. 13.
Knockout and complementation analysis of
S. aureus cap5E, cap5F, and
cap5G. Colony immunoblot analysis with polyclonal
antiserum to CP5. A, Newman CP5+. B,
8325-4 CP5 (mutation in cap5E). C,
8325-4 (pCAP16). D, strain G01 (ermB in
cap5G). E, GO1 (pKOR2). F, G01
(pLI50). G, strain F4 (ermB in cap5F).
H, F4 (pCAP17). I, F4 (pCAP16).
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DISCUSSION |
This report describes for the first time the biosynthesis steps
from the precursor UDP-D-GlcNAc to a nucleotide precursor sugar that is consistent with UDP-L-FucNAc; five distinct
enzymatic steps, catalyzed by three enzymes, are sufficient. The
deduced amino acid sequences of the gene products from P. aeruginosa and S. aureus are highly homologous. WbjB
and Cap5E show 67% identity and 81% similarity over 332 amino acids.
Similarly, WbjC and Cap5F share 40% identity and 59% similarity over
370 amino acids. The last enzymes in the pathway, WbjD and Cap5G, were
also highly homologous, with 57% identity and 71% similarity over 377 amino acids.
Two putative pathways for the biosynthesis of UDP-L-FucNAc
have been proposed in the literature, both involving
UDP-D-ManNAc as the precursor (15, 20). Hypothesizing
putative pathways based solely on in silico analysis is
prone to error, because occasionally the primary protein sequences of
enzymes are highly homologous even though they catalyze completely
different conversions. This is especially true for SDR proteins, which
include dehydratases, dehydrogenases, epimerases, isomerases, and
reductases. Members of this family play crucial roles in the
biosynthesis of nucleotide-activated sugars throughout prokaryotes and
eukaryotes, in steroid biosynthesis, and in alcohol metabolism (40).
Typically, they have the nucleotide-binding motif
GXXGXXG or GXXXGXG near the
N terminus that is essential for coenzyme binding (41). Moreover, they
all share a motif YXXXK, the active site. This motif can be
extended to the so-called catalytic triad SYK. Recently, SMK has been
described as a slight modification of this triad in WbpM from P. aeruginosa, an enzyme that is putatively involved in
UDP-N-acetyl-D-fucosamine/quinovosamine biosynthesis from UDP-D-GlcNAc (42). FlaA1 from
Helicobacter pylori, catalyzing the same reaction as WbpM,
has an SYK catalytic triad (45). Interestingly, of the two SDR proteins
involved in UDP-L-FucNAc-biosynthesis, one has SMK
(WbjB/Cap5E) and the other has SYK (WbjC/Cap5F). WbjB/Cap5E,
WbjC/Cap5F, and WbpM/FlaA1 are highly homologous and yet catalyze
distinct reaction steps in nucleotide-activated sugar biosynthesis.
Thus, it is not surprising that UDP-L-FucNAc biosynthesis
follows a pathway different from those proposed previously by Jiang
et al. (15) and Lee and Lee, respectively (20). Limitations
of the proposals have been taken into account by these research groups.
The most efficient way to analyze biochemical pathways is through
enzymatic assays. For the third enzyme involved in
UDP-L-FucNAc biosynthesis, it was easier to propose a
putative function based solely on its significant homologies to
UDP-D-GlcNAc 2-epimerases, which do not belong to the
diverse SDR protein family.
Our results showed that WbjB/Cap5E has at least two functions with both
4,6-dehydratase and 5-epimerase activities. Most likely, it also
catalyzes epimerization at C-3. Bifunctionality within the SDR protein
family also was reported for Gmd from Aneurinibacillus thermoaerophilus, which has been described as
4,6-dehydratase/4-reductase converting GDP-D-mannose to
GDP-D-rhamnose (46), and for hFX, a
3,5-epimerase/4-reductase involved in the biosynthesis of
GDP-L-fucose (47). The first reaction step catalyzed by
WbjB/Cap5E is 4,6-dehydration, which appears to be quantitative,
because CE analysis revealed no UDP-D-GlcNAc left on
conversion. The second reaction step of WbjB/Cap5E is an equilibrium
reaction, as would be expected for an epimerase reaction.
Using CE, coupled CE-MS, and NMR analyses, we were able to show the
reaction cascade that converts UDP-D-GlcNAc into a product that is consistent with UDP-L-FucNAc. CE analysis of the
WbjB/Cap5E-catalyzed reaction showed a quantitative conversion of
UDP-D-GlcNAc to two new nucleotide-activated sugars. To
investigate these two peaks further, coupled CE-MS was performed.
Surprisingly, MS showed a mixture of two peaks with
m/z ratios of 588 and 606. A 4,6-dehydration is
supposed to result in loss of water and thus a peak at
m/z 588 only. Two peaks could correspond to a
bifunctional enzyme with both 4,6-dehydratase and 3,5-epimerase
activity. Time course experiments with CE-MS/MS/MS clearly demonstrated
that the peak at m/z 606 is not
UDP-D-GlcNAc. Loss of a fragment ion of
m/z 138 indicated the removal of the hydroxyl
group at C-6, because a
30 shift (generation of formaldehyde) is
characteristic of hexoses. Thus, water has been added to the hexose
moiety after 4,6-dehydration. There are two possibilities for this
scenario: formation of a geminal diol at the keto-group at C-4 or ring
opening to result a nucleotide-activated sugar in the open form.
Because the final assignments could not be accomplished using an MS
approach, we decided to purify the two putative keto-intermediates for
NMR analysis, despite their expected instability (46, 48, 49). The
purification of the intermediate compound, which migrated faster than
NADP+ during CE analysis, was achieved by anion exchange
chromatography. The compound proved to be unstable, partially
decomposing to the monosaccharide and UDP, but its structure could be
elucidated using nanoprobe NMR. One-dimensional TOCSY and
one-dimensional TOCSY-NOESY allowed assignment of the hydrogens on C-1,
C-2, C-3, C-5, and C-6, as well as on the acetyl group. Lack of a
hydrogen at C-4 and lack of a second 13C signal in the
keto-range indicated the presence of a ketal at C-4, thus excluding the
possibility of an open sugar. The quaternary carbon could be assigned
by HMBC. The other carbons were assigned with HSQC. Thus, the structure
of this intermediate sugar is
UDP-2-acetamido-2,6-dideoxy-
-D-xylo-4-hexulose, resulting from 4,6-dehydration of UDP-D-GlcNAc. Anion
exchange chromatography did not yield a second compound. Therefore, we concentrated a WbjB reaction mix and, after addition of
D2O, analyzed this mixture by NMR spectroscopy. Using
similar selective techniques as with the other intermediate compound,
we were able to assign a second structure,
UDP-2-acetamido-2,6-dideoxy-
-L-arabino-4-hexulose, which is formed by 5-epimerization of
UDP-2-acetamido-2,6-dideoxy-
-D-xylo-4-hexulose. These data suggested that WbjB/Cap5E most likely has 4,6-dehydratase, 5-epimerase, and 3-epimerase activities. The lack of the expected final
product of this reaction cascade, namely
UDP-2-acetamido-2,6-dideoxy-
-L-lyxo-4-hexulose, may be due to the highly unstable lyxo-product or an
equilibrium favoring the first two intermediates,
UDP-2-acetamido-2,6-dideoxy-
-D-xylo-4-hexulose and
UDP-2-acetamido-2,6-dideoxy-
-L-arabino-4-hexulose.
An alternative possibility would be a bifunctional enzyme
WbjC/Cap5F with both 3-epimerase/ 4-reductase activities. However,
because we are not able to purify either
UDP-2-acetamido-2,6-dideoxy-
-L-arabino-4-hexulose or
UDP-2-acetamido-2,6-dideoxy-
-L-lyxo-4-hexulose,
we are not able to investigate this scenario further.
The next reaction step in the biosynthesis of UDP-L-FucNAc
involves the conversion of the intermediate sugars into a new compound that comigrated with the precursor UDP-D-GlcNAc in CE
analysis. NADH as well as NADPH could be used as hydride donor. CE-MS
analysis showed a new peak at m/z 590, which
would be expected after reduction of the keto-intermediate. CE-MS/MS
experiments provided further evidence that the reduction was on the
hexose moiety of the nucleotide-activated sugars. To complete
identification of this compound, we purified the reduction product
using anion exchange chromatography. The sugar was concentrated and,
after addition of D2O, was analyzed by NMR. Unlike the
unstable keto-intermediates, this sugar turned out to be highly stable.
One-dimensional TOCSY, one-dimensional NOESY, and other techniques
demonstrated that the WbjC/Cap5F reduction product is
UDP-2-acetamido-2,6-dideoxy-
-L-talose, sometimes
referred to as UDP-L-pneumosamine or
UDP-L-N-acetylpneumosamine (PneNAc) (50). Interestingly, the CP of S. pneumoniae type 5 is
composed not only of D-glucose and D-glucuronic
acid but also of
2-acetamido-2,6-dideoxy-D-xylo-4-hexulose, L-PneNAc, and L-FucNAc (50). Thus, three of
the intermediates and final products of the pathway described in the
present study are part of the type 5 capsular polysaccharide.
Unfortunately, no nucleotide sequence data of S. pneumoniae
type 5 biosynthesis locus are yet available. Based on the biochemical
characterization of the Wbj and Cap proteins, one can predict that
homologs of WbjB/Cap5E, WbjC/Cap5F, and WbjD/Cap5G will be present in
the corresponding S. pneumoniae type 5 gene cluster.
The last step of the UDP-L-FucNAc biosynthesis pathway
would require C-2-epimerization of
UDP-2-acetamido-2,6-dideoxy-
-L-talose. WbjD/Cap5G is
moderately homologous to the S. aureus
UDP-D-GlcNAc 2-epimerase (Cap5P). As expected, WbjD/Cap5G
catalyzed UDP-2-acetamido-2,6-dideoxy-
-L-talose to
produce a new sugar peak, as detected by CE analysis. Further experiments have been performed using CE-MS. The fragmentation pattern
did not change on this reaction from the one obtained for
UDP-2-acetamido-2,6-dideoxy-
-L-talose, which is
consistent with epimerization. As mentioned above,
UDP-2-acetamido-2,6-dideoxy-
-L-talose proved to be a
very stable sugar, unlike the final reaction product. When purified
UDP-2-acetamido-2,6-dideoxy-
-L-talose was incubated with
WbjD, UDP was produced by decomposition of the final product UDP-L-FucNAc to the monosaccharide and the nucleotide as
observed by CE. Based on this experience and the following reasons,
we were not able to pursue NMR analysis of UDP-L-FucNAc,
the putative product of the WbjD reaction. First, the unstable nature
of this sugar nucleotide posed difficulty in its preparation. Second, the epimerization activity of WbjD yielded a mixture of UDP-TalNAc and
UDP-Fuc2NAc. Our CE (Fig. 2F) and CE-MS-MS results (Fig.
3D) support these findings. The proportion of UDP-Fuc2NAc
was small in the mixture, thus making it difficult to purify and
resolve the two compounds. Another piece of evidence to support our
interpretation that WbjD and its homolog Cap5G in S. aureus
are C2-epimerases is the fact that L-FucNAc is a common
sugar residue shared by the O-polysaccharide of P. aeruginosa O11 and the type 5 capsular polysaccharide of S. aureus. Therefore, once we were able to prove that UDP-TalNAc is
formed by the activity of WbjC/Cap5F, we clearly correlated the
activities of the first two enzymes to the modification of C-3, C-4,
C-5, and C-6 of the substrate, UDP-GlcNAc. The only logical reaction to
follow is C-2-epimerization to produce UDP-Fuc2NAc, and our data
obtained from CE and CE-MS-MS support this interpretation.
It was intriguing to observe that UDP-L-FucNAc acted as an
inhibitor of WbjB/Cap5E. When all of the enzymes were added
simultaneously, more NADPH was left in the reaction mixture than when
the enzymes were added sequentially, allowing each step to go to
completion. CE-MS clearly demonstrated that some
UDP-D-GlcNAc was still left after the simultaneous
reaction. No UDP-D-GlcNAc could be detected when the
reactions were done consecutively. Feedback inhibition is a common way
to regulate nucleotide-activated sugar biosynthesis, as in
GDP-L-fucose biosynthesis, in which the final product,
GDP-
-L-fucose, acts as an inhibitor of Gmd (51).
The cofactor dependence of WbjB/Cap5E proved to be crucial. The absence
of any added cofactor or the addition of NAD+ instead of
NADP+ resulted in almost no conversion at all. The
conversion observed (<5%) was most likely due to NADP+
bound to the enzyme even during protein purification. In contrast, WbjC/Cap5F used both NADH and NADPH as hydride donors in the reduction reaction. Because of the lack of commercially available substrate, kinetic parameters have not been investigated. 4-Keto-sugars have frequently been described in the literature as unstable (46, 48, 49).
Our report confirms these studies, because the keto-intermediates involved in this pathway readily decompose to the corresponding monosaccharides and UDP. Because the first enzyme acting in the pathway
is multifunctional, a coupled assay using both WbjB/Cap5E and
WbjC/Cap5F would not result in proper kinetic data of the WbjC/Cap5F-catalyzed reduction reaction. However, such coupling could
be a convenient means to at least qualitatively screen for inhibitors
of WbjB/Cap5E and WbjC/Cap5F by measuring the decrease of NADH or NADPH
using A340 nm. The use of both hydride donors
has also been described for Gmd and Rmd in GDP-D-rhamnose biosynthesis (46) as well as for RmlD in dTDP-L-rhamnose
biosynthesis (48).
The construction of a knockout mutant of the first gene
(wbjB) involved in the biosynthesis of
UDP-L-FucNAc clearly demonstrated that the corresponding
enzyme WbjB is unmistakably involved in the biosynthesis of B-band O
antigen in P. aeruginosa serotype O11. Inactivation of
wbjB resulted in complete loss of B-band O antigen, as
detected by silver staining of an SDS-PAGE resolved LPS and by Western
immunoblotting analysis with polyclonal or monoclonal antibodies to
serotype O11 (Fig. 12, A-C). The banding pattern of LPS
isolated from the mutant BK103B was due to A-band O antigen, as was
proven by the use of mAb N1F10 specific for A-band O antigen (Fig. 12,
A and D). The P. aeruginosa gene
wbjB and its S. aureus homolog
cap5E were able to restore production of B-band O antigen
fully, when provided on an E. coli/P. aeruginosa shuttle
vector pUCP27. The three cap5 genes cap5E,
cap5F, and cap5G are all required for capsule
synthesis in S. aureus. In this study, the insertional
inactivation of cap5F and cap5G confirmed the
essential role of these genes in CP5 production. A previous study had
identified that a naturally occurring mutation in strain 8325-4 could
be mapped to cap5E (39). However, it is apparent only from
analysis of the recently available genomic sequence that this mutation
arose from a single amino acid change (Met134 to
Arg134) in the SMK domain, underlining the important
catalytic role of this triad. The data described here represent a
"proof of principle" that the genes coding for the enzymes
catalyzing the biosynthesis of UDP-L-FucNAc are conserved
among Gram-positive and Gram-negative bacteria and can functionally
cross-complement each other fully.
In conclusion, we propose a novel biosynthetic pathway for
UDP-L-FucNAc, which is the putative precursor of
L-FucNAc in both Gram-positive and Gram-negative bacteria.
The data in this study suggest the respective function of the three
proteins to be: (i) WbjB/Cap5E is a trifunctional
UDP-D-GlcNAc 4,6-dehydratase/5-epimerase/3-epimerase, (ii)
WbjC/Cap5F is a
UDP-2-acetamido-2,6-dideoxy-
-L-lyxo-4-hexulose 4-reductase, and (iii) WbjD/Cap5G is likely a
UDP-2-acetamido-2,6-dideoxy-
-L-talose 2-epimerase. The
enzymes involved are promising targets in the search for new broad
spectrum antibiotics. Work is under way to develop assays suitable to
screen compound libraries for potential inhibitors.