From the
Zentrum für Ultrastrukturforschung
und Ludwig Boltzmann-Institut für Molekulare Nanotechnologie,
Universität für Bodenkultur Wien, A-1180 Wien, Austria and
Institut für Chemie, Universität
für Bodenkultur Wien, A-1190 Wien, Austria
Received for publication, January 27, 2003 , and in revised form, May 9, 2003.
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ABSTRACT |
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INTRODUCTION |
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The repeating unit of the glycan chain of Aneurinibacillus
thermoaerophilus L420-91T
(8,
9), a member of the
Bacillus/Clostridium group, consists of the sugars
D-rhamnose and 3-acetamido-3,6-dideoxy-D-galactose
(3-N-acetylfucosamine (D-Fucp3NAc)). The backbone
of each repeating unit is built by four D-rhamnose units, and two
of them are substituted by -1,2-linked D-Fucp3NAc
units (10,
11). Both constituting sugars
are well known components of the LPS of Gram-negative bacteria
(1214).
Derivatives of D-Fucp3N have also been found in the LPS of
clinical strains of Vibrio cholerae
(15) and Acinetobacter
baumannii (16), where the
acetyl group is replaced by modified amino acids and acyl residues,
respectively.
Recently, we were able to characterize the biosynthesis of the GDP-D-rhamnose, one of the sugar components of the glycan chain of A. thermoaerophilus L420-91T (17). The other constituent, D-Fucp3NAc, was first described in cell-free extracts of Xanthomonas campestris by Volk and Ashwell (18). They proposed that D-glucose will eventually be converted into dTDP-D-Fucp3NAc by amidation with L-glutamate and pyridoxal 5-phosphate (PLP) as co-factor followed by acetylation with acetyl-CoA (18). This proposal was further substantiated by Shibaev (19), who proposed a model for the biosynthesis of nucleotide-activated D-Fucp3NAc. Two of the enzymes presumably involved in dTDP-D-Fucp3NAc biosynthesis were first identified in the dTDP-L-rhamnose pathway (for review, see Ref. 20). These are glucose-1-phosphate thymidyltransferase (RmlA) and dTDP-D-glucose-4,6-dehydratase (RmlB), which catalyze the reaction of glucose 1-phosphate and thymidine triphosphate to dTDP-D-glucose and in the second step to dTDP-6-deoxy-D-xylohex-4-ulose. This latter compound serves as substrate for a series of reactions, including the formation of dTDP-D-Fucp3NAc (21). Activities of RmlA and RmlB in crude extracts of A. thermoaerophilus L420-91T indicated a possible involvement of these two enzymes in the biosynthesis of dTDP-D-Fucp3NAc (22). Recently, the gene cluster responsible for LPS biosynthesis of X. campestris has been identified (14). Downstream of the dTDP-L-rhamnose genes the authors described a cluster of 15 genes to be involved in the biosynthesis of the LPS glycan chain of X. campestris. Two genes of the cluster, namely wxcM and wxcK, were assumed to code for enzymes that synthesize dTDP-D-Fucp3NAc. WxcM was proposed to be bifunctional and to catalyze both the isomerization and the transacetylation reaction. The second ORF, WxcK, was assumed to be responsible for the transamination reaction. So far none of the described genes has been functionally expressed nor have the gene products been biochemically characterized.
This report is the first functional characterization of the biosynthesis pathway of dTDP-D-Fucp3NAc in the S-layer glycoprotein glycan of the Gram-positive thermophile A. thermoaerophilus L420-91T. We could show that five enzymes, including a novel type of isomerase, are involved in the biosynthesis of this nucleotide-activated precursor of D-Fucp3NAc.
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EXPERIMENTAL PROCEDURES |
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Bacterial Strains and Culture ConditionsA. thermoaerophilus
L420-91T was grown in SVIII media at 55 °C
(10). Escherichia
coli DH5 (K-12 F
80d
lacZ
M15 endA1 recA1 hsdR17
(rK
mK) supE44 thi-1 gyrA96 relA1
(lacZYA-argF) U169) (Invitrogen) was used for cloning
purposes. Enzyme overexpression was performed in E. coli BL21(DE3)
(F ompT hsdSB
(rB mB) gal
dcm (DE3)). For selective growth ampicillin (Sigma) and kanamycin
(Invitrogen) were used at a concentration of 50 µg/ml.
DNA Manipulations, Polymerase Chain Reaction, and Gene
IdentificationAll standard DNA manipulation and transformation
procedures were performed according to the methods described by Sambrook and
Russell (23) or according to
the protocols recommended by the specific manufacturers. The gene cluster was
identified by chromosome walking as described previously
(24). Briefly, restriction
cuts of chromosomal DNA of A. thermoaerophilus L420-91T
were ligated into pBluescript II SK(+) from Stratagene (Amsterdam, The
Netherlands), and PCR was performed with forward primers of the known
sequences and reverse primers of the plasmid. Primers were designed with attB1
and attB2 sites for the insertion into the GatewayTM vector pDonr201
(Invitrogen). E. coli DH5 was transformed with the vectors by
electroporation with a Gene Pulser apparatus (Bio-Rad). The vectors were
prepared with a Miniprep kit of Qiagen (Hilden, Germany) and sequenced by
AGOWA GmbH (Berlin, Germany). PCR was performed with a PCR Sprint thermocylcer
from Hybaid (Ashford, UK). The nucleotide sequences were analyzed by the
sequence analysis program OMIGATM (Accelrys, Unterhaching-München,
Germany). Homology searches and sequence aligments were performed with the
BLAST tool at NCBI (25) and
Multialin (26),
respectively.
Plasmid ConstructionFrom the data obtained by DNA sequencing, the open reading frames of interest were amplified by PCR. The primers used in these reactions were designed with attB1 and attB2 for the insertion into the vectors of the GATEWAYTM system (Invitrogen). The following primers were employed: gFdtA_for1, 5'-attB1-GCATGGAAAATAAAGTTATTAACTTCAAGAA-3'; gFdtA_rev1, 5'-attB2-CTCTACAATTGCGTTAGGGTG-3'; gFdtB_for1, 5'-attB1-GCATGATTCCTTTTTTGGATTTA-3'; gFdtB_rev1, 5'-attB2-CAAATTTTGCAACCTTGG-3'; gFdtC_for1, 5'-attB1-GCATGTCTAGTTCTAGTGAAACTT-3'; gFdtC_ rev1, 5'-attB2-AAAAGGAATCATCATACCCCA-3'.
PCR fragments flanked by the attB1 and attB2 sites can be directionally
recombined into pDonr201 in one simple step and subsequently selected for. The
positive clones obtained were used to insert the genes into the expression
vectors pDest15 for GST fusions or pDest17 for (His)6 fusions. For
plasmid propagation E. coli DH5 was used. The expression
vectors gFdtA, gFdtB, and gFdtC were finally transformed by electroporation
into E. coli BL21(DE3). The exact protocol for these procedures was
obtained directly from the manufacturer. All plasmid constructs were sequenced
for sequence verification.
Overexpression and Purification of Recombinant
ProteinsCells carrying the plasmids gFdtA, gFdtB, and gFdtC were
grown in 0.7 liters of culture volumes of Luria Bertani medium containing 50
µg/ml ampicillin to an optical density at 600 nm of 0.6. Expression was
induced with 1 mM
isopropyl-1-thio--D-galactopyranoside for 3 h. The cells were
harvested, washed in 20 mM phosphate buffer, pH 7.4, and
resuspended in 10 ml of the same buffer. Cells were lysed on ice by
ultrasonication, and cell debris was removed by ultracentrifugation at 331,000
x g. Protein purification was performed as recommended by the
manufacturer by applying the supernatant either to a HiTrap chelating column
using Ni2+ as the metal ion or a GSTrap column,
dependent on the tag used. After dialysis against 20 mM Tris/HCl
buffer, pH 7.7, the enzymes were further purified by anion exchange
chromatography on a Mono Q HR 5/5 column with 0.5 M KCl as the
eluent. Fractions containing the enzymes, as determined by SDS-PAGE
(27), were concentrated with
centrifugal filter devices Ultrafree-MC (cutoff 10,000 Da; Millipore, Vienna,
Austria) and stored at 4 °C or 20 °C. In both cases 50%
glycerol and 1 mM 1,4-dithio-DL-threitol were used as
stabilizers. Protein concentrations were determined according to Bradford
(28) using the Bio-Rad protein
assay (Bio-Rad).
Enzyme Assays and Substrate SynthesisAll standard assays for the determination of enzyme functions contained either 50 nmol of dTDP-6-deoxy-D-xylohex-4-ulose or dTDP-D-Fucp3N and were performed in 50 mM K2HPO4 buffer, pH 7.4, containing 5 mM MgCl2. The assay for the conversion of dTDP-6-deoxy-D-xylohex-4-ulose to dTDP-D-Fucp3N contained 5 nmol of FdtB, 5 nmol PLP, and 50 nmol of L-glutamate and was performed with and without the addition of FdtA. To test the function of the transacetylase 5 nmol of FdtC was incubated with 50 nmol of acetyl-CoA and 50 nmol of dTDP-D-Fucp3N. All assays were performed at 37 °C and analyzed by reverse phase (RP)-HPLC with 0.5 M NaH2PO4 solution buffer, pH 6.0, as the mobile phase at a flow rate of 0.6 ml/min (29). The eluate was monitored with a UV detector at 254 nm. Approximately 50 mg of dTDP-6-deoxy-D-xylohex-4-ulose were synthesized as described previously (30). To synthesize dTDP-D-Fucp3N, 20 µmol of dTDP-6-deoxy-D-xylohex-4-ulose, 1 µmol of PLP, 50 µmol of L-glutamate, 200 nmol of FdtA, and 200 nmol of FdtB were incubated in 5 ml of 50 mM K2HPO4 buffer, pH 7.4, and 5 mM MgCl2 at 37 °C for 1 h. The volume was reduced by vacuum evaporation to approximately 1 ml, and the material was purified by RP-HPLC with 0.2 M triethylammonium acetate buffer, pH 6.0, as the mobile phase. To remove the HPLC buffer the solution containing dTDP-D-Fucp3N was frozen and lyophilized. An overall yield of 80% of dTDP-D-Fucp3N could be achieved.
Kinetic MeasurementsTo determine the kinetic constants of FdtC the reaction between the free sulfhydryl group of SH-CoA, a reaction product during the transacetylation, and Ellman's reagent (5,5'-dithio-bis(2-nitrobenzoic acid) (31) was measured continuously as the increase in absorbance at 412 nm on a Hitachi U-2010 (Maidenhead, UK) spectrophotometer. The resulting enzymatic activities were calculated with a molar absorption coefficient of 14,150 M1 cm1 according to the instructions of the manufacturer. A standard assay was performed in a 50 mM potassium phosphate buffer, pH 7.4, containing 5 mM MgCl2, 200 µM 5,5'-dithio-bis(2-nitrobenzoic acid, 25200 µM dTDP-D-Fucp3N, 600 µM acetyl-CoA, and 3 nM FdtC. To determine kinetic constants for acetyl-CoA the same assay was used, but the acetyl-CoA concentration was varied from 25 to 200 µM, and the dTDP-D-Fucp3N concentration was kept constant at 600 µM. To obtain the apparent Michaelis constant, Km, kinetic data were fitted directly to the Michaelis-Menten equation by using the program Sigma Plot (SPSS Science, Chicago, IL). Vmax and the final enzyme concentration E were used to determine the turnover number kcat of the substrates employed (30).
NMR MeasurementsSpectra were recorded at 300 K at 300.13
MHz for 1H and at 75.47 MHz for 13C with a Bruker AVANCE
300 spectrometer equipped with a 5-mm quadrupole nuclear probe head with
z gradients. Data acquisition and processing were performed with the
standard XWINNMR software (Bruker). 1H spectra were referenced to
2,2-dimethyl-2-silapentane-5-sulfonic acid ( = 0), 13C
spectra were referenced externally to 1,4-dioxane (
= 67.40), and
31P spectra were recorded at 121.50 MHz and referenced externally
to H3PO4 (
= 0). 1H/13C and
1H/31P heteronuclear multiple quantum coherence spectra
and 1H/13C heteronuclear multiple bond correlation
spectra were recorded in the phase-sensitive mode using time-proportional
phase increments and pulsed field gradients for coherence selection. Spectra
resulted from a 256 x 4096 data matrix, with 1800 or 800 scans per t1
value, respectively. For in situ experiments of the isomerase
reaction, two samples containing dTDP-6-deoxy-D-xylohex-4-ulose (9
mg) in 20 mM K2HPO4 buffer (in 0.6 ml of
D2O, pH 7.4) were simultaneously prepared and kept at 310 K. After
adding FdtA (0.113 mg) and mixing in the NMR vial, NMR measurements were
immediately started and recorded at distinct time intervals. To disprove a
bifunctional activity of FdtB, the same experiment was performed with the
transaminase. Briefly, 0.1 mg of FdtB was incubated with
dTDP-6-deoxy-D-xylohex-4-ulose (9 mg) in phosphate buffer, pH 7.4,
at 310 K.
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RESULTS |
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For the newly identified genes we suggest the following names: fdtA for the putative isomerase, fdtB for the transaminase, and fdtC for the transacetylase. In this designation f codes for fucosamine, d for the D configuration and t (= three) indicates the substituted C-3 atom of the sugar ring. Based on the results from our work in A. thermoaerophilus L420-91T (22) and also from previous studies on X. campestris (14, 18, 36), we propose a general biosynthesis pathway for D-Fucp3NAc as depicted in Fig. 2.
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Overexpression and Purification of FdtA, FdtB, and FdtC Before overexpression and purification of the enzymes the sequences of the corresponding plasmids containing the open reading frames of interest were verified by nucleotide sequencing. To confirm the function proposed for the three enzymes involved in the biosynthesis of dTDP-D-Fucp3NAc they were heterologously overexpressed in E. coli and purified by affinity chromatography and anion exchange chromatography. To overcome problems with protein stability, FdtA and FdtC were expressed with a GST tag and purified using GSTrap columns. In addition, FdtA was also expressed as N-terminal (His)6-tagged fusion protein as was FdtB. Both recombinant proteins could be highly purified on a HiTrap chelating column (Ni2+). To improve the purity of the enzymes an anion exchange chromatography step on a Mono Q column was applied subsequently. Expression and purification was monitored by SDS-PAGE and showed that the apparent molecular mass of all proteins was in agreement with the values deduced from the nucleotide sequences (Fig. 3). To increase the stability, the enzymes were concentrated by ultrafiltration before storage at 4 °C or 20 °C and supplemented with 50% glycerol. The concentrated enzyme preparations were stable at 4 °C for several months with only a marginal decrease of their enzyme activity (data not shown).
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FdtA Is Responsible for the Isomerization of dTDP-6-deoxy-D-xylohex-4-ulose into dTDP-6-deoxy-D-xylohex-3-uloseThis conversion is supposed to be the essential step in the formation of dTDP-D-Fucp3NAc in vivo. However, it was not possible to isolate this intermediate product by HPLC methods, including reverse phase and ion exchange chromatography. RP-HPLC analysis of dTDP-6-deoxy-D-xylohex-4-ulose showed an elution profile identical to that described in a recent work (35). The isomerase product dTDP-6-deoxy-D-xylohex-3-ulose revealed an identical elution profile, and thus, distinction from the 4-keto educt was impossible (Fig. 4B). The activity of FdtA was monitored by a simultaneous incubation with dTDP-6-deoxy-D-xylohex-4-ulose with FdtB and the required co-substrates, as described under "Experimental Procedures." The resulting product, dTDP-D-Fucp3N, was detectable by HPLC analysis (Fig. 4C). The activities of FdtA as GST- and His-tagged proteins were also examined, and both fusion proteins showed comparable activities.
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The direct conversion of dTDP-6-deoxy-D-xylohex-4-hexulose into
the 3-keto product by the action of FdtA was studied by NMR spectroscopy.
Before the interpretation of the NMR data, a full spectral assignment of the
starting material was performed. It matched favorably with the literature data
(37) and indicated a
4.5:1 ratio of hydrate to keto form of
dTDP-6-deoxy-D-xylohex-4-ulose. H,H-COSY spectroscopy revealed a
clear connectivity from H-1 to H-2 and H-3 for both forms, thus eliminating
the occurrence of 3-keto precursors within the detection limits. Analysis of
the incubation product of FdtA with dTDP-6-deoxy-D-xylohex-4-ulose
at pD 7.4 indicated the formation of
dTDP-6-deoxy-D-xylohex-3-ulose. As shown in
Fig. 5 conversion into the
3-keto form occurred rapidly; however, a full assignment could not be achieved
due to the small signal intensity and severe signal overlap of the remaining
substrate. Comparison of the data with a non-enzymatic control reaction and
assignment of signals separated from the bulk region allowed a partial
assignment of the spectra. Whereas in the non-catalyzed reaction no
significant conversion was seen after 2 h, the NMR spectrum of the enzymatic
reaction performed at 37 °C already indicated after 5 min the formation of
a new component (Fig. 5). The
anomeric proton of this reaction product was observed at lower field (5.86
ppm) with a 3JH-1,P coupling constant of 7.0 Hz
and a 3JH-1,H-2 coupling constant of 4.5 Hz.
The signal of H-2 at 4.93 ppm gave a coupling to the anomeric phosphate but no
further spin-spin coupling to position 3, which in conjunction with the
observed downfield shift would be consistent with the presence of a
neighboring keto group at C-3. Moreover, an additional 6-deoxy signal was
observed at 1.31 ppm (3JH-6,H-5 6.5 Hz), which
was shown by COSY and heteronuclear multiple quantum coherence experiments to
be correlated to H-5/C-5 signals at 4.46 ppm/66.6 ppm, respectively,
indicating a change of configuration at the neighboring C-4 carbon (relative
to dTDP-glucose). These data are also distinctly different from those
published for dTDP-6-deoxy-D-ribohex-3-ulose, obtained from a
reversed transamination reaction
(38) and by a non-enzymatic
conversion of substrate (37).
The combined evidence would, thus, be in agreement with an inversion of
configuration at C-4. Based on the integration values of the anomeric protons
of the intermediates, the proportion of the 3-keto product reached a maximum
of
1011% after 1 h and then slowly decreased due to formation of
additional products that were not further analyzed.
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FdtB Aminates Specifically dTDP-6-deoxy-D-xylohex-3-ulose to Form dTDP-D-Fucp3NBecause only limited amounts of dTDP-6-deoxy-D-xylohex-3-ulose were formed in the FdtA reaction the formation of dTDP-D-Fucp3N was first performed in a simultaneous incubation of dTDP-6-deoxy-D-xylohex-4-ulose with FdtA and FdtB. FdtB requires PLP and L-glutamate as co-factors. Divalent metal ions are not required, which is in accordance with previous studies on transaminases (39). Among several amino group donors tested, including L-glutamine, L-alanine, and L-aspartate, only L-glutamate resulted in a turnover. The product of the amination reaction was monitored by RP-HPLC (Fig. 4C) and was subsequently purified as described under "Experimental Procedures."
To obtain better insight into the isomerization reaction and the specific
activity of FdtB the 4-keto substrate was incubated without the addition of
FdtA. Incubation of the reaction mixture at pH 7.4 in the absence of
co-factors/co-substrates was monitored by NMR spectroscopy. The spectra
recorded in a time course of 24 h did not show differences to a non-enzymatic
control reaction (compare with Fig.
5A). Incubation of 4-keto substrate with FdtB and the
co-factors yielded over a time span of 36 h 30% conversion to
dTDP-D-Fucp3N.
To test the substrate specificity of the transaminase further, the isomerase reaction was conducted for 7 h before the addition of FdtB, which would also allow the onset of nonspecific isomerization reactions. Work-up of the reaction mixture and NMR analysis revealed a single product, dTDP-D-Fucp3N, similar to the experiments where both enzymes had been added to the substrate simultaneously.
The structure of the product generated by the action of the enzymes FdtA/FdtB could be fully assigned on the basis of the NMR spectroscopic data (Table II, substance V). Thus, for the 3-amino-3-deoxy-derivative dTDP-D-Fucp3N, the signal at 53.4 ppm was due to a nitrogen-bearing carbon atom being correlated to a H-3 signal, displaying a large trans-diaxial coupling constant 3JH-2,H-3 (11.1 Hz) and a small value for the coupling constant to H-4 (3JH-3,H-4 3.2 Hz). This is consistent with a 3-amino-3,6-dideoxy-D-xylo configuration. In addition, the 13C NMR signals of C-4 and C-5 experienced an up-field shift to 68.9 and 68.3 ppm, respectively.
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FdtC Catalyzes the Transfer of an Acetyl Group to dTDP-D-Fucp3N to Form dTDP-D-Fucp3NAcThe transacetylation reaction represents the last step of the formation of nucleotide-activated D-Fucp3NAc. The commonly used acetyl donor of these reactions acetyl-CoA was also used in this experiment. Isolation of the final product was achieved by RP-HPLC (Fig. 4D), as described under "Experimental Procedures," and this product was also characterized by NMR spectroscopy (Table II, substance VI). The product of the N-acetylation reaction displayed signals of an N-acetyl group at 2.02 ppm/22.8 ppm for the 1H and 13C signals of the methyl group and signals of the pyranose unit that were in good agreement with the data of D-Fucp3NAc units observed in the native polysaccharide (10). Furthermore, an 1H/31P-correlated spectrum confirmed the presence of the diphosphoryl unit in dTDP-D-Fucp3NAc.
Because of the fact that only couple assays were possible for the isomerase and transaminase reactions, no kinetic constants were determined. However, reaction kinetics were analyzed for the transacetylation reaction. For the determination of the kinetic constants of FdtC with acetyl-CoA and dTDP-D-Fucp3N Ellman's reagent was used. Both substrates exhibited comparable affinity to FdtC. The Michaelis-Menten constants Km were 66.7 (±15.9) and 61.0 (±4.4) µM for dTDP-D-Fucp3N and acetyl-CoA, respectively. The turnover numbers, kcat, were 2.3 (± 0.2) and 3.1 (± 0.1) s1.
General Properties of the EnzymesAll purified enzymes
showed maximum activities between 50 and 55 °C, although some activity
could also be detected at 30 °C. FdtB was also shown catalyze the reverse
reaction using -ketoglutarate and dTDP-D-Fucp3N as
the substrate instead of L-glutamate and
dTDP-6-deoxy-D-xylohex-3-ulose. By adding FdtA to the reaction
mixture the equilibrium shifted toward
dTDP-6-deoxy-D-xylohex-4-ulose. The reverse reaction was monitored
by HPLC (data not shown). However, a reverse reaction catalyzed by FdtC could
not be detected.
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DISCUSSION |
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In a previous report the gene cluster and the enzymes involved in the biosynthesis of GDP-D-rhamnose in A. thermoaerophilus L420-91T have been identified and characterized biochemically (17). Chromosome walking revealed the presence of five open reading frames downstream of the GDP-D-rhamnose operon, presumably responsible for the biosynthesis of dTDP-D-Fucp3NAc. Two of the ORFs showed high similarities to rmlA and rmlB, and two others were homologous to the protein families of transaminases and transacetylases. The fifth gene in the cluster showed homology with a putative enzyme, which is assumed to catalyze potentially an isomerase reaction (14). To characterize the enzymes involved in the biosynthetic pathway they were expressed in E. coli and purified.
FdtA catalyzes the isomerization step in the biosynthesis pathway of dTDP-D-Fucp3NAc. In this reaction the 4-keto intermediate is isomerized to the 3-keto product, with D-xylo configuration. Several attempts to isolate the reaction product of the isomerase reaction dTDP-6-deoxy-D-xylohex-3-ulose by HPLC were unsuccessful. Thus, to confirm the function of this enzyme, on-line NMR measurements were applied. These experiments showed that FdtA catalyzes the rapid formation of dTDP-6-deoxy-D-xylohex-3-ulose in small amounts. This finding points toward the importance of a tightly controlled product formation in organisms such as X. campestris, which besides dTDP-D-Fucp3NAc also synthesizes dTDP-L-rhamnose from the same source of precursor. Here we provide the first functional description of a specific isomerase that catalyzes the transition of dTDP-6-deoxy-D-xylohex-4-ulose into dTDP-6-deoxy-D-xylohex-3-ulose.
FdtA shares no homologies with known isomerase families, and no common motifs could be detected. Because the enzyme lacks metal ions and oxido reduction co-factor binding motifs, the isomerization reaction catalyzed by FdtA may act by a novel mechanism. We identified gene homologs of fdtA in several Gram-negative organisms, although their specific functions are not known yet. Because of the importance of the isomerase reaction step, the presence of FdtA may imply that these organisms are able to synthesize dTDP-D-Fucp3N (14) or dTDP-3-acetamido-3,6-dideoxy-D-glucose derivatives (43). Almost all gene clusters described for these organisms also contain in the vicinity of fdtA either transaminases, transacetylases, or rml genes (Table I). Multiple sequence alignment of FdtA with 11 sequences found in the data base yielded remarkable homologies, indicating the presence of potential reaction or binding domains (Fig. 6). Further investigations will be required to identify the reaction centers and the amino acids involved in the conversion reaction. Because of the inability to transform A. thermoaerophilus L420-91T genetically so far, we were not able to create an fdtA-deficient mutant for complementation experiments. Previous attempts to create knock-out mutants of wxcM, the fdtA-like open reading frame in X. campestris, were also not successful (14).
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FdtA occurs in A. thermoaerophilus L420-91T as a single functional enzyme, whereas in X. campestris it is part of a bifunctional enzyme also catalyzing the acetylation reaction (14). The presence of bifunctional enzymes in these pathways seems to be a common characteristic of Gram-negative organisms, whereas in Gram-positive organisms the use of single functional genes is preferred (44). Recently, Perelle et al. (43) have described a new enzyme, WbsB, which presumably is involved in the biosynthesis of the acylated quinovosamine residue ([R]-3-hydroxybutyramido-3-acetamido-3,6-dideoxy-D-glucose) occurring in the O-antigen of E. coli O91, which shows high homology to FdtA (Fig. 6). These data imply that the transition of the keto group from C-4 to C-3 of the sugar ring may follow a similar pathway, albeit resulting in the formation of the 6-deoxy-D-ribo configuration.
FdtB is a monofunctional and highly specific enzyme that catalyzes the transamination of dTDP-6-deoxy-D-xylohex-3-ulose to dTDP-D-Fucp3N. It does not possess any isomerase activity; however, it may convert catalytic amounts of non-enzymatically formed 3-keto substrate present in the reaction mixture. Previously it had been suggested that the formation of dTDP-6-deoxy-D-ribohex-3-ulose could be achieved chemically via enolization without the addition of an isomerase (37). For an efficient turnover of the substrate, however, the presence of FdtA is indispensable. Our experiments indicated that incubation of dTDP-6-deoxy-D-xylohex-4-ulose with FdtB and the corresponding co-substrates at pH 7.4 leads over time only to dTDP-D-Fucp3N.
FdtB turned out to be a member of the DegT/DnrJ/EryC1/StrS family of aminotransferases that contains among others 3-amino-5-hydrobenzoic acid synthase, an enzyme required for the biosynthesis of ansamycin antibiotics. Crystal structure analysis of this enzyme revealed the presence of a PLP binding site, which is conserved among the aspartate aminotransferase structural family and is also present in FdtB. This specific site is characterized by a serine residue that is followed by three to five residues before the active site lysine (SX35K) (45). In the BLAST search the amino acid sequence around the PLP binding site revealed a highly conserved region, obviously a common feature of aminotransferases, which use nucleotide-activated 6-deoxy sugars as substrate. Besides the DNA sequences described in Table I, similar transferases may also be involved in the biosynthesis of several antibiotics that contain C-3-aminated 6-deoxy sugars, such as oleandomycin (46), daunomycin (47), erythromycin (48), and tylosin (32). The formation of C-3 aminated 6-deoxy sugars also requires the presence of an isomerase-like enzyme, which until today was found only in the biosynthetic pathway of tylosin of S. fradiae (49).
The nucleotide-activated form of D-Fucp3N may also play an important role in the formation of acetylated sugar precursors as described in this study but also in other acylated carbohydrates. In several Gram-negative organisms the corresponding LPS was found to contain D-Fucp3N acylated with e.g. (R,R)-3-hydroxy-3-methyl-5-oxoproline such as in V. cholerae O5 (15) or L-2-acetoxypropionamido (50) and D-3-hydroxybutyryl groups (16) in A. baumannii. The biosynthesis of these modified D-Fucp3N units may, therefore, have similar if not identical pathways. However, because of the lack of genetic data and functional characterization of these pathways this assumption remains to be further substantiated.
FdtC catalyzes the transfer of an acetyl group to
dTDP-D-Fucp3N. It is the last enzyme in this reaction
sequence and contains a hexapeptide repeat sequence motif, a common feature of
a number of bacterial and plant acetyltransferases. This motif is
characterized by imperfect, tandem-repeated copies of the six amino acids
(L/I/V)(G/A/E/D)X2(S/T/A/V)X
(51). Recently, x-ray crystal
structure analysis of N-acetylglucosamine 1-phosphate
uridyltransferase, a bifunctional enzyme whose C-terminal domain is
responsible for the CoA-dependent acetylation of glucose 1-phosphate in E.
coli, revealed that the left-handed parallel -helix formed by the
hexapeptide repeat sequence provides the pocket for the binding of the sugar
substrate and catalyzes the transfer of the acetyl group
(52). Only for this last step
in the biosynthesis of dTDP-D-Fucp3NAc were we able to get
kinetic data. The low turnover number obtained may be caused by temperatures
below the optimum temperature of that enzyme. These data are in agreement with
previous kinetic studies on the biosynthesis of dTDP-L-rhamnose
(22). Because of the fact that
we were not able to purify the 3-keto substrate for the transamination
reaction, reliable kinetic data could neither be obtained for the isomerase
nor the transaminase reaction.
In summary, in this work we were able to elucidate the biosynthetic pathway of dTDP-D-Fucp3NAc. This precursor is involved in the synthesis of glycan chains of glycosylated S-layer proteins as well as of LPS O-antigens. The biosynthesis involves, in addition to a transaminase and a transacetylase, a highly specific isomerase. Sequence comparison of the involved genes suggests that the pathway may also be valid in Gram-negative organisms, such as in X. campestris (14). A. thermoaerophilus L420-91T is a moderately thermophilic organism with optimal growth temperatures in the range of 5055 °C. In this context it is interesting to note that enzymes from thermophilic organisms, such as the Rml enzymes from A. thermoaerophilus DSM 10155, have shown a higher thermal stability than similar enzymes from mesophilic organisms (22). Therefore, exploration of their potential biotechnological application is definitely worthwhile.
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FOOTNOTES |
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* This work was supported by the Austrian Science Fund Grant P14209
[GenBank]
-MOB (to
P. M.). The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
¶ To whom correspondence should be addressed: Zentrum für Ultrastrukturforschung und Ludwig Boltzmann-Institut für Molekulare Nanotechnologie, Universität für Bodenkultur Wien, Gregor-Mendel-Strasse 33, A-1180 Wien, Austria. Tel.: 43-1-47654-2202; Fax: 43-1-4789112; E-mail: paul.messner{at}boku.ac.at.
1 The abbreviations used are: S-layer, surface layer; LPS,
lipopolysaccharide; GST, glutathione S-transferase; PLP,
pyridoxal-5-phosphate; ORF, open reading frame; RP, reverse phase; HPLC, high
performance liquid chromatography; D-Fucp3NAc,
3-acetamido-3,6-dideoxy-D-galactose.
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ACKNOWLEDGMENTS |
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REFERENCES |
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