From the Department of Preventive Dentistry and the
¶ Department of Prosthetic Dentistry I, Kyushu University Faculty
of Dentistry, Fukuoka 812-8582, Japan
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ABSTRACT |
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The serotype-specific polysaccharide antigen of
Actinobacillus actinomycetemcomitans Y4 (serotype b)
consists of D-fucose and L-rhamnose. Thymidine
diphosphate (dTDP)-D-fucose is the activated nucleotide
sugar form of D-fucose, which has been identified as a
constituent of structural polysaccharides in only a few bacteria. In
this paper, we show that three dTDP-D-fucose synthetic
enzymes are encoded by genes in the gene cluster responsible for the
synthesis of serotype b-specific polysaccharide in A. actinomycetemcomitans. The first and second steps of the
dTDP-D-fucose synthetic pathway are catalyzed by
D-glucose-1-phosphate thymidylyltransferase and dTDP-D-glucose 4,6-dehydratase, which are encoded by
rmlA and rmlB in the gene cluster,
respectively. These two reactions are common to the well studied
dTDP-L-rhamnose synthetic pathway. However, the enzyme
catalyzing the last step of the dTDP-D-fucose synthetic
pathway has never been reported. We identified the fcd gene
encoding a dTDP-4-keto-6-deoxy-D-glucose reductase. After purifying the three enzymes, their enzymatic activities were analyzed by reversed-phase high performance liquid chromatography. In addition, nuclear magnetic resonance analysis and gas-liquid chromatography analysis proved that the fcd gene product converts
dTDP-4-keto-6-deoxy-D-glucose to dTDP-D-fucose.
Moreover, kinetic analysis of the enzyme indicated that the
Km values for
dTDP-4-keto-6-deoxy-D-glucose and NADPH are 97.3 and 28.7 µM, respectively, and that the enzyme follows the
sequential mechanism. This paper is the first report on the
dTDP-D-fucose synthetic pathway and
dTDP-4-keto-6-deoxy-D-glucose reductase.
Capsular polysaccharides are the outermost structures on a
bacterial cell and play a critical role in the interactions of the cell
with the environment (1). These molecules are prominent, structurally
and serologically diverse antigens that are involved in pathogenic
processes and in mediating resistance to important host defense
mechanisms. In the case of human pathogens, a large number of different
capsule serotypes have been identified (1). Previously, we reported
that the serotype b-specific antigen of Actinobacillus
actinomycetemcomitans Y4 is a capsular polysaccharide-like antigen consisting of two deoxyhexoses, L-rhamnose and
D-fucose (2).
A. actinomycetemcomitans is a nonmotile, Gram-negative,
capnophilic, fermentative coccobacillus that has been implicated in the
etiology and pathogenesis of localized juvenile periodontitis (3-5),
adult periodontitis (6), and severe nonoral human infections (7).
A. actinomycetemcomitans strains isolated from the human oral cavity are divided into five serotypes, a, b, c, d, and e (8-10).
The serologic specificity is defined by the polysaccharide on the
surface of this organism (11), and the serotype-specific antigen is one
of the immunodominant antigens in the organism (12, 13).
In general, L-rhamnose is a component of many bacterial
polysaccharides (14). The synthesis of thymidine diphosphate
(dTDP)1-L-rhamnose,
which is the activated nucleotide sugar form of L-rhamnose, has been well studied by several investigators (15-18). The anabolism of dTDP-L-rhamnose from D-glucose-1-phosphate
is catalyzed by four enzymes, namely, D-glucose-1-phosphate
thymidylyltransferase, dTDP-D-glucose 4,6-dehydratase,
dTDP-4-keto-6-deoxy-D-glucose 3,5-epimerase, and
dTDP-4-keto-L-rhamnose reductase (Fig.
1) (15-18). On the other hand,
D-fucose has been identified as a component of other
bacterial polysaccharides in only a few publications (19, 20), and
there is no report concerning the activated nucleotide sugar form of
D-fucose in the synthesis of dTDP-D-fucose. On
the basis of the chemical structure of dTDP-D-fucose,
Shibaev (21) predicted that dTDP-D-fucose synthesis occurs
along the same pathway that leads to dTDP-L-rhamnose but
that it branches off after the intermediate
dTDP-4-keto-6-deoxy-D-glucose, when the intermediate is
acted on by a reductase to produce dTDP-D-fucose (Fig. 1).
This hypothesis, however, is not supported by independent evidence.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Pathways for the synthesis of
dTDP-L-rhamnose and dTDP-D-fucose from
D-glucose-1-phosphate and dTTP. The genes encoding the
enzymes are indicated in parentheses.
Recently, a large gene cluster associated with the biosynthesis of the
serotype-specific polysaccharide antigen of serotype b A. actinomycetemcomitans was cloned and characterized by our group
(22). It follows from the components of serotype b-specific antigen
that some genes coding for enzymes responsible for the synthesis of
dTDP-D-fucose may be located in the cloned gene cluster. This is the first report to identify genes coding for essential enzymes
involved in the dTDP-D-fucose synthetic pathway and to characterize the purified enzymes.
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EXPERIMENTAL PROCEDURES |
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Bacterial Strains and Culture Conditions--
Escherichia
coli DH5 (supE44
lacU169 (
80
lacZ
M15) hsdR17 recA1 endA1 gyrA96 thi-1
relA1) (23) was used in the DNA manipulations. E. coli
SØ874 (lacZ2286 trp-49
(sbcB-rfb)86
upp-12 relA1 rpsL150
) (24), which was
kindly provided by M. K. B. Berlyn (E. coli Genetic Stock Center, Department of Biology, Yale University, New
Haven, CT), was used as the host strain to express genes inserted into
pHSG399 (25), pGEM-T vector (Promega, Madison, WI), or pTrc99A (26).
E. coli ER2566 (F
fhuA2 (lon) ompT lacZ::T7 gene1 gal
sulA11
(mcrC-mrr) 114::IS10 R(mcr-73::miniTn10-TetS)2
R(zgb-210::Tn10)(TetS)
endA1 (dcm)) (New England Biolabs, Beverly, MA)
was grown as a host strain when the IMPACT T7 One-Step Protein
Purification System (New England Biolabs) was used. E. coli
strains were grown aerobically in 2× TY broth at 30 or 37 °C (23).
When required, antibiotics were used to supplement media at the
indicated concentrations: ampicillin at 50 µg/ml and chloramphenicol
at 20 µg/ml.
DNA Manipulation-- DNA isolation, restriction endonuclease digestion, ligation, and transformation were performed essentially as described by Sambrook et al. (23).
Plasmid Construction--
Plasmid pARF400 was constructed from
pARF211 containing the region responsible for the synthesis of serotype
b-specific polysaccharide antigen (22). A 7.0-kilobase
NheI-BamHI fragment of pARF211 was ligated to
XbaI-BamHI-digested pHSG399 (25) to produce
pARF400. Seven deletion derivatives of pARF400 were constructed by
digestion of the plasmid with appropriate restriction enzymes (Fig.
2A). These eight plasmids were
used to detect the gene coding for a dTDP-4-keto-6-deoxy-D-glucose reductase.
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The DNA fragments carrying rmlA, rmlB, rmlC, or rmlD of A. actinomycetemcomitans were amplified by PCR using pARF100 (22) as a template and the following oligonucleotides as primers: for the rmlA gene, 5'-ATGAAGGGTATTATTCTT-3' and 5'-TTATTTCTCCTCGTTGAT-3'; for the rmlB gene, 5'-ATGTTGAAAACTATTTTA-3' and 5'-TTATTGACTGCCTAAACG-3'; for the rmlC gene, 5'-TTAAAGCGTATGCGGAGT-3' and 5'-TTAAAATTTTACCGTTTC-3'; and for the rmlD gene, 5'-ATCAACGAGGAGAAATAA-3' and 5'-TTTACTCCGCATACGCTT-3'. Each PCR product was cloned separately into pGEM-T vector (Promega) to make four plasmids. These plasmids were subsequently digested with NcoI and SalI and subcloned into NcoI-SalI-digested pTrc99A (26) to yield pARF405, pARF401, pARF406, and pARF411.
To construct pTYB2 derivatives, the rmlA, rmlB, and fcd genes were amplified by PCR from pARF100 (22). The following sets of primers were used for amplification: for the rmlA gene, 5'-ATGCATATGAAGGGTATTATTCTT-3' and 5'-GGGCCCGGGTTTCTCCTCGTTGATTAA-3'; for the rmlB gene, 5'-ATGCATATGTTGAAAACTATTTTA-3' and 5'-GGGCCCGGGTTGACTGCCTAAACGCTC-3'; and for the fcd gene, 5'-ATGCATATGATTATCGGAAATGGA-3' and 5'-GGGCCCGGGTTTAATAGCATAATATTT-3'. These primers were designed so that NdeI and SmaI restriction sites (underlined) were created in the PCR product. Each PCR product was purified using a QIAquick PCR Purification Kit (Qiagen, Chatsworth, CA) and, after double digestion with NdeI and SmaI, directly ligated to an NdeI-SmaI double-digested pTYB2 vector to produce pARF514, pARF509, and pARF517.
Preparation of Crude Enzyme Extracts--
Crude enzyme extracts
were obtained from E. coli SØ874 containing each plasmid
(pARF405, pARF401, pARF406, pARF411, pARF400, pARF420, pARF421,
pARF422, pARF423, pARF424, pARF425, and pARF426) as described
previously (27). The resulting crude enzyme extracts were stored at
20 °C until used.
Purification of Enzymes--
The rmlA,
rmlB, and fcd products were purified using the
expression vector, pTYB2 (New England Biolabs). To purify the
rmlA, rmlB, and fcd products, E. coli
ER2566 was transformed with pARF514, pARF509, and pARF517,
respectively. Optimally, cells were grown in 500 ml of 2× TY broth
with ampicillin at 30 °C to an optical density of 0.7 at 600 nm.
Cultures were induced with 1 mM
isopropyl--thiogalactopyranoside. The cells were harvested 4 h
after induction and lysed by ultrasonication. The cell extracts were
obtained by centrifugation at 12,000 × g for 30 min at
4 °C. Binding to chitin beads (New England Biolabs), cleavage of the
fusion protein (in 20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 0.1 mM EDTA, and 30 mM
dithiothreitol at 4 °C in 20 h), and elution of each product
was done according to the manufacturer's instructions. The protein
fraction was collected, and glycerol was added to a final concentration
of 50%. The purified proteins were stored at
20 °C until used.
The purity of the proteins was checked by SDS-PAGE.
Enzyme Assay--
The overall function of the rmlA,
rmlB, rmlC, and rmlD products was
ascertained by the detection of rhamnose. Moreover, the gene coding for
a dTDP-4-keto-6-deoxy-D-glucose reductase was identified by
the production of fucose. The reaction mixture (20 µl), containing 50 mM Tris-HCl (pH 7.6), 12 mM MgCl2,
4 mM -D-glucose-1-phosphate, 4 mM dTTP, 0.45 unit of inorganic pyrophosphatase (Roche
Molecular Biochemicals), 6 mM NADPH, and appropriate
amounts of the crude enzyme extracts, was incubated at 37 °C for
3 h. The solution was mixed with 180 µl of 0.5 M
HCl. After the tube was sealed under vacuum, the mixed solution was
heated at 80 °C for 1 h to hydrolyze the reaction products.
The assay for the conversion of D-glucose-1-phosphate and
dTTP to dTDP-D-glucose,
dTDP-4-keto-6-deoxy-D-glucose, or dTDP-D-fucose was performed by reversed-phase HPLC. The incubation mixture (40 µl)
contained 50 mM Tris-HCl (pH 7.6), 12 mM
MgCl2, 4 mM
-D-glucose-1-phosphate, 4 mM dTTP, 0.9 unit
of inorganic pyrophosphatase, 8 mM NADPH, and 2 µg of the
purified proteins/ml. Incubations were carried out at 37 °C for
3 h.
Identification of Products-- Sugar components in the hydrolyzed solution were coupled with 2-aminopyridine, and the pyridylamino sugars were analyzed by HPLC with an anion-exchange column (PALPAK type A column; Takara, Kyoto, Japan) as described previously (27, 28). The identification of dTTP, dTDP-D-glucose, dTDP-4-keto-6-deoxy-D-glucose, or dTDP-D-fucose was confirmed using reversed-phase HPLC as described by Tonetti et al. (29). Samples (10 µl) diluted 10-fold with distilled water were injected onto a TSKgel ODS-80Ts column (0.46 by 15 cm; Tosoh, Tokyo, Japan) with 0.5 M KH2PO4 as the mobile phase at a flow rate of 1.0 ml/min at 40 °C. The eluate was monitored with a UV detector at 260 nm.
NMR Analysis--
The fractions containing
dTDP-D-fucose were collected from the reaction mixture
using the HPLC method described above. The reaction mixture (4 ml)
contained 50 mM Tris-HCl (pH 7.6), 12 mM
MgCl2, 4 mM
-D-glucose-1-phosphate, 4 mM dTTP, 90 units
of inorganic pyrophosphatase, 8 mM NADPH, and 2 µg of the
purified rmlA, rmlB, and fcd products/ml. To
remove the KH2PO4, four volumes of ethanol were
added to the solution collected. The mixture was then centrifuged at
12,000 × g for 10 min. After ethanol precipitation, the supernatant (60 ml) was concentrated to approximately 1 ml by
evaporation and desalted by gel filtration on a Sephadex G-10 column
(1.5 × 69 cm; flow rate 30 ml/h; Amersham Pharmacia Biotech Inc.)
with distilled water as the eluent. The sample was lyophilized and
redissolved in 0.5 ml of D2O and transferred to a 5-mm NMR tube. 1H NMR analysis was performed on a Bruker AM400
spectrometer. The measurement was made at 298 K. The chemical shifts
were referenced to 3-trimethylsilylproionate-D4 at 0.0 ppm.
The 1H spectrum of 128 scans was recorded with
presaturation of HOD resonance at 4.72 ppm.
Gas-Liquid Chromatography Analysis-- dTDP-D-Fucose as a sample was obtained by the same method as described under "NMR Analysis." About 2 mg of dTDP-D-fucose was dissolved in 300 µl of 0.1 M HCl. An ampoule containing the solution was sealed under vacuum and heated at 80 °C for 1 h to hydrolyze dTDP-D-fucose. The solution was then evaporated to remove water and HCl. The pellet, D-fucose, or L-fucose was converted into the corresponding D-(+)-2-octylglycoside acetate by the method of Leontein et al. (30). Each product was characterized by gas-liquid chromatography (model GC-14B; Shimadzu Works, Tokyo, Japan) with a fused silica capillary column (CP Sil-88, 0.25 mm by 50 m; Chrompack Inc., Bridgewater, NJ) at 200 °C. Approximately 1 µl of the sample was injected, and the split ratio was 1:20. Helium was used as a carrier gas at a flow rate of 0.9 ml/min.
Kinetics--
At first,
dTDP-4-keto-6-deoxy-D-glucose was synthesized as a
substrate and purified. The reaction mixture (20 ml) contained 50 mM Tris-HCl (pH 7.6), 12 mM MgCl2,
4 mM -D-glucose-1-phosphate, 4 mM dTTP, 450 units of inorganic pyrophosphatase, and 2 µg
of the purified rmlA and rmlB products/ml. The
dTDP-4-keto-6-deoxy-D-glucose in the reaction mixture was
purified by HPLC, ethanol precipitation, and gel filtration as
described above.
The general approach for determining the kinetic properties of
dTDP-4-keto-6-deoxy-D-glucose reductase is as described by Cleland (31) and Segel (32). The experiments were designed to provide
data by varying the concentration of
dTDP-4-keto-6-deoxy-D-glucose at several fixed
concentrations of NADPH. The enzyme assay was carried out in a
microcuvette containing 0.5 ml of a reaction mixture composed of 50 mM Tris-HCl (pH 7.6), 12 mM MgCl2,
80 ng of the purified protein/ml, 18.8-300 µM of
dTDP-4-keto-6-deoxy-D-glucose, and 25-150 µM
of NADPH. The kinetic constants were determined by the estimating the
amount of NADPH oxidation during the conversion of
dTDP-4-keto-6-deoxy-D-glucose to dTDP-D-fucose.
The rate of the decrease in optical density at 340 nm was determined spectrophotometrically.
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RESULTS |
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Identification of the ORFs Involved in the Conversion of D-Glucose-1-phosphate into dTDP-L-Rhamnose-- Previously, we reported cloning and characterizing a large gene cluster associated with the biosynthesis of the serotype-specific polysaccharide antigen of A. actinomycetemcomitans Y4 (serotype b) and that the gene cluster contains 24 ORFs (22). ORF7, ORF6, ORF9, and ORF8, whose gene products exhibit strong homologies to the rmlA, rmlB, rmlC, and rmlD products, respectively (22), were subcloned into the expression vector, pTrc99A. The resulting plasmids were introduced into E. coli SØ874, which is a rough mutant lacking the E. coli rml gene cluster, and the gene products were extracted from the transformants of strain SØ874. dTDP-L-Rhamnose was synthesized from D-glucose-1-phosphate, dTTP, and NADPH only when all four crude enzyme extracts were added to the reaction mixture (data not shown). This result as well as the high homologies of the ORF7, ORF6, ORF9, and ORF8 products to the rmlA, rmlB, rmlC, and rmlD gene products, respectively, suggests that ORF7, ORF6, ORF9, and ORF8 encode D-glucose-1-phosphate thymidylyltransferase, dTDP-D-glucose 4,6-dehydratase, dTDP-4-keto-6-deoxy-D-glucose 3,5-epimerase, and dTDP-4-keto-L-rhamnose reductase, respectively. Therefore, ORF7, ORF6, ORF9, and ORF8 have been renamed rmlA, rmlB, rmlC, and rmlD, respectively.
Identification of the Gene Encoding dTDP-4-Keto-6-deoxy-D-glucose Reductase-- To identify the gene encoding dTDP-4-keto-6-deoxy-D-glucose reductase, eight deletion derivatives of pARF211 (pARF400, pARF420, pARF421, pARF422, pARF423, pARF424, pARF425, and pARF426) (Fig. 2A) were constructed, and a crude enzyme extract from E. coli SØ874 harboring each plasmid was obtained. Each crude enzyme extract was added to a reaction mixture containing D-glucose-1-phosphate and dTTP as substrates and crude enzyme extracts from E. coli SØ874 expressing rmlA and rmlB as enzymes. Fucose was not detected in the hydrolysate of the reaction mixture containing the crude enzyme extract from E. coli transformant harboring pARF425 by HPLC after being coupled with 2-aminopyridine (Fig. 2B, top). The HPLC analysis using the crude enzyme extract from E. coli transformant harboring pARF421, pARF422, or pARF424 instead of pARF425 exhibited a similar pattern (data not shown). On the other hand, pyridylaminated fucose was detected in the hydrolysate of the reaction mixture containing the crude enzyme extract from E. coli transformant harboring pARF400 by HPLC (Fig. 2B, bottom). A similar HPLC profile was exhibited when using the crude enzyme extract from E. coli transformant harboring pARF420, pARF423, or pARF426 instead of pARF400 (data not shown). To determine relative retention times, pyridylaminated xylose was added to each sample as an internal standard. These results suggested that dTDP-4-keto-6-deoxy-D-glucose reductase is encoded by ORF14. Fucose was not detected in the reaction mixtures lacking one of the crude enzyme extracts from E. coli SØ874 expressing rmlA, rmlB, or ORF14. We therefore renamed ORF14 fcd according to the new gene nomenclature of Reeves et al. (33). These results were summarized in Fig. 2A.
Purification and Characterization of Enzymes Involved in the
Synthesis of dTDP-D-Fucose--
To unequivocally determine
the function of the rmlA, rmlB, and
fcd products, these proteins were purified by affinity
chromatography as described in detail under "Experimental
Procedures." The proteins were homogeneous as judged by SDS-PAGE
(Fig. 3). The molecular masses of the
denatured polypeptides determined by SDS-PAGE of 32, 39, and 27 kDa
agree well with the predicted molecular masses of the rmlA,
rmlB, and fcd gene products, respectively.
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An in vitro assay was carried out with these purified
enzymes. The conversion of D-glucose-1-phosphate and dTTP
to dTDP-D-glucose was observed in the reaction catalyzed by
the rmlA product (Fig. 4B).
dTDP-4-Keto-6-deoxy-D-glucose was detected as a broad peak in the reaction mixture containing the purified products of both rmlA and rmlB as enzymes (Fig. 4C).
The reason why the peak of this intermediate is apparently broad and
poor is unknown. dTDP-D-Fucose was detected in the reaction
mixture containing all three purified proteins, the rmlA,
rmlB, and fcd products (Fig. 4D). The
retention time of the dTDP-D-glucose formed was compared
with that obtained for a commercially available standard. However, a
standard for dTDP-4-keto-6-deoxy-D-glucose could not be
purchased from any company. Therefore, the standard for
dTDP-4-keto-6-deoxy-D-glucose was synthesized using the
purified rmlA and rmlB products of E. coli K-12 (34). 1H NMR analysis and gas-liquid
chromatography analysis were performed to confirm the structure and
configuration of this final product. NADPH was not detected under the
HPLC condition used.
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NMR Analysis of dTDP-D-Fucose--
Approximately 2 mg
of sugar nucleotide were pooled from several HPLC runs on an ODS-80Ts
(Fig. 4E). After removing the excess phosphate by adding
ethanol, the solution was concentrated by evaporation and further
purified with a Sephadex G-10 column equilibrated with demineralized
water. The fractions containing sugar nucleotide were pooled,
lyophilized, and dissolved in D2O. The NMR spectra of
D-fucose, dTDP, and this sample are shown in Fig.
5. The signals of D-fucose
are rather complicated because of and
anomers coexisting (Fig.
5A). Each peak is assigned to
6 (1.24 ppm),
6 (1.28 ppm),
2 (3.46 ppm),
3 (3.66 ppm),
4 (3.77 ppm),
2 (3.79 ppm),
4 (3.83 ppm),
5 (3.83 ppm),
3 (3.89 ppm),
5 (4.22 ppm),
1 (4.58 ppm), and
1 (5.21 ppm) from the analogy to
L-fucose (35). Comparing those peaks with the sample
spectrum (Fig. 5C) and considering the splitting patterns,
the peaks from D-fucose in Fig. 5C are assigned
as follows;
6 (1.24 ppm, doublet),
2 (3.77 ppm, double doublet),
4 (3.85 ppm, double doublet),
3 (3.94 ppm, double doublet),
5
(4.31 ppm, quartet), and
1 (5.59 ppm, broad). From the observed
coupling constants (J(1,2) = 2.44 Hz,
J(2,3) = 10.76 Hz, J(3,4) = 2.96 Hz, J(4,5) = 2.96 Hz), the orientations
of H1, H2, H3, H4, and H5 are equatorial, axial, axial, equatorial, and
axial, respectively. Thus the integrity of
-D-fucose
structure in the current product was confirmed. The broadening and
large downfield shift (0.4 ppm) of the H1 band can be attributed to
neighboring phosphate groups, which also affects the H2 band, thereby
causing the poor resolution. Comparing with dTDP (Fig. 5B),
other peaks are assigned to protons on the nucleotide moiety as
follows: CH3 (1.95 ppm, singlet), H6 (7.77 ppm, singlet),
H1' (6.37 ppm, triplet), H2' (2.38 ppm, double doublet), H3' + H5'
(4.19 ppm, overlapped), and H4' (4.65 ppm, multiplet). The signal of
H4' is rather small, perhaps because it is affected by decoupling of
water nearby.
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Determination of the Absolute Configuration of the Fucosyl Residue
in the Final Product--
On the basis of the structure and
configuration of dTDP-4-keto-6-deoxy-D-glucose, we can
predict that the absolute configuration of the fucosyl residue in
dTDP-fucose is D. However, the prediction was not supported
by direct evidence. Gas-liquid chromatography analysis was performed to
prove the hypothesis. After dTDP-fucose was hydrolyzed, fucosyl residue
in dTDP-fucose was detected as a D-(+)-2-octylglycoside
acetate. For this fucosyl residue, four peaks were obtained, the two
pyranosides and the two furanosides. Relative retention times of four
peaks obtained from fucosyl residue in dTDP-fucose agreed with those
obtained not from L-fucose but from D-fucose
(Fig. 6). Thus the D absolute
configuration of the fucose in dTDP-fucose was determined.
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Kinetic Characterization of
dTDP-4-Keto-6-deoxy-D-glucose
Reductase--
dTDP-4-Keto-6-deoxy-D-glucose was
synthesized and purified as a substrate. The concentration of
dTDP-4-keto-6-deoxy-D-glucose was calculated from the
absorbance at 320 nm in 0.1 M NaOH assuming a molar
absorption coefficient of 5,600 M1·cm
1 (18).
Double reciprocal plots of the initial velocity as a function either of
dTDP-4-keto-6-deoxy-D-glucose concentration at different NADPH concentrations or of NADPH concentration at different
dTDP-4-keto-6-deoxy-D-glucose concentrations yielded
apparently nonparallel lines (Fig.
7A). This pattern indicates a
sequential mechanism. From the secondary plots (Fig. 7, B
and C), the Km values for
dTDP-4-keto-6-deoxy-D-glucose and NADPH and the
Vmax value (turnover number) were estimated to
be 97.3 µM, 28.7 µM, and 7.74 µM·min1, respectively. Because the
concentration of the enzyme added to the reaction mixture was 2.97 nM, the Kcat value was estimated to
be 2.61 × 103 min
1.
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DISCUSSION |
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Previously, we identified a gene cluster associated with the biosynthesis of the serotype-specific polysaccharide antigen consisting of L-rhamnose and D-fucose from A. actinomycetemcomitans Y4 (serotype b) (22). D-Fucose is a component of some bacterial polysaccharides (19, 20). This sugar, however, is not widespread in nature. The biosynthetic precursor of D-fucose is believed to be dTDP-D-fucose, but little attempt has been made to isolate the enzymes involved in the conversion of D-glucose-1-phosphate and dTTP to dTDP-D-fucose or the genes coding for the enzymes. On the other hand, the synthesis of dTDP-L-rhamnose has been investigated extensively (15-18). The dTDP-L-rhamnose synthetic pathway consists of four enzymes catalyzing the reaction sequence from D-glucose-1-phosphate and dTTP to dTDP-L-rhamnose through three intermediates, and the rmlA, rmlB, rmlC, and rmlD genes coding for the four enzymes have been identified (24, 34, 36-38) (Fig. 1). In this paper, we identified the genes required for the synthesis of dTDP-D-fucose and dTDP-L-rhamnose and showed that dTDP-D-fucose was formed by the reduction of dTDP-4-keto-6-deoxy-D-glucose, an intermediate in the dTDP-L-rhamnose biosynthetic pathway, at C-4 (Fig. 1).
We initially identified the four genes required for the synthesis of dTDP-L-rhamnose in A. actinomycetemcomitans. As expected from the amino acid sequences of their products, ORF7, ORF6, ORF9, and ORF8 were suggested to encode D-glucose-1-phosphate thymidylyltransferase, dTDP-D-glucose 4,6-dehydratase, dTDP-4-keto-6-deoxy-D-glucose 3,5-epimerase, and dTDP-4-keto-L-rhamnose reductase, respectively. On the other hand, the identity of dTDP-4-keto-6-deoxy-D-glucose reductase, which forms dTDP-D-fucose, was not predicted from amino acid sequence homology. Instead, the dTDP-D-fucose synthesis gene was located by detecting fucose coupled with 2-aminopyridine in the reaction mixture containing crude extracts from E. coli strains harboring rmlA, rmlB, and one of the eight deletion plasmids (Fig. 2). This experiment led us to conclude that the ORF14 gene encodes a dTDP-4-keto-6-deoxy-D-glucose reductase. We renamed ORF14 the fcd gene. The amino acid sequence deduced from the fcd gene showed an identity of <10% with previously reported proteins in a homology search using the program FASTA. However, a BLAST search showed that the amino acid sequence of the fcd gene product shared limited homology with that of CDP-abequose synthase of Salmonella enterica LT2 (39). CDP-Abequose synthase catalyzes a similar reaction, in which the keto group on carbon 4 of a hexosyl group is attacked. The difference between the two reductases lies in their substrates. dTDP-4-Keto-6-deoxy-D-glucose is converted to the L-manno configuration by dTDP-4-keto-6-deoxy-D-glucose 3,5-epimerase and is then reduced by dTDP-4-keto-L-rhamnose reductase, the rmlD product, to form dTDP-L-rhamnose (Fig. 1). The fcd product did not share homology either with the amino acid sequence of the rmlD product or with those of any previously reported NDP-6-deoxyhexosyl-4-ulose reductases except CDP-abequose synthase of S. enterica. An NADPH binding motif GXGXXA (40) was found in the N-terminal region in the fcd product but not in the corresponding region of CDP-abequose synthase.
HPLC analysis of dTDP-D-fucose formed by the purified
enzymes showed that D-glucose-1-phosphate was converted to
dTDP-D-glucose by the rmlA product, and this was
then converted into dTDP-4-keto-6-deoxy-D-glucose by the
rmlB product. These reactions are common to the first two steps of the dTDP-L-rhamnose biosynthetic pathway (Fig. 1).
The retention times of the sugar nucleotide formed by the
rmlA product and that of its derivative converted by the
rmlB product agreed with those of dTDP-D-glucose
formed by purified glucose-1-phosphate thymidylyltransferase from
E. coli DH5 and dTDP-4-keto-6-deoxy-D-glucose formed by E. coli dTDP-D-glucose
4,6-dehydratase, respectively (data not shown). In addition,
dTDP-4-keto-6-deoxy-D-glucose formed by E. coli
glucose-1-phosphate thymidylyltransferase and
dTDP-D-glucose 4,6-dehydratase was converted to a sugar
nucleotide that was eluted at the same retention time as that of
dTDP-D-fucose produced by the three A. actinomycetemcomitans enzymes. These results also support the
prediction that the rmlA and rmlB products of
A. actinomycetemcomitans Y4 are glucose-1-phosphate
thymidylyltransferase and dTDP-D-glucose 4,6-dehydratase,
respectively, and that the fcd product reduces dTDP-4-keto-6-deoxy-D-glucose to form
dTDP-D-fucose.
dTDP-4-Keto-6-deoxy-D-glucose reductase catalyzes a bimolecular group reduction reaction. The mechanisms of such reactions are divided into two major categories, namely, the sequential and ping-pong mechanisms, based on the order of interaction between the enzyme and its substrates (31, 32). In contrast to the ping-pong mechanism, in the sequential mechanism all the substrates must combine to form a ternary complex before the product is formed. Our kinetic studies of NADPH and dTDP-4-keto-6-deoxy-D-glucose are consistent with its action by a sequential mechanism (Fig. 7A). The Km and Vmax values cannot be compared with those of other similar enzymes because no other NDP-6-deoxyhexosyl-4-ulose reductases have been characterized.
It is very interesting to note that the primary structure of dTDP-4-keto-6-deoxy-D-glucose reductase from A. actinomycetemcomitans Y4 exhibits little homology with other NDP-6-deoxyhexosyl-4-ulose reductases when considering its origin and evolution. There are only a few reports on D-fucose as a component of bacterial polysaccharide. Furthermore, of the five serotype antigens (a-e) of A. actinomycetemcomitans strains, only the serotype b antigen contains D-fucose. Moreover, Southern blot analysis indicated that the fcd gene is specific to serotype b (data not shown). It is, however, unlikely that this gene evolved in A. actinomycetemcomitans Y4. The G+C content (37.7%) of the operon essential for producing serotype-specific antigen is lower than the average G+C content (45.6%) of A. actinomycetemcomitans Y4 chromosomal DNA (22). This low G+C content suggests that the gene cluster was transferred from another species. A lower G+C content has been found in many gene clusters involved in the synthesis of various bacterial polysaccharides (34, 37, 41, 42). In particular, the region containing ORF14-ORF19 has the lowest G+C content (27.0%), and the discrepancy of the G+C content within the gene cluster suggests that the fcd and dTDP-L-rhamnose synthetic genes have different origins. In addition, the amino acid sequences of the proteins encoded by genes around the fcd gene are not homologous with any reported proteins. These findings suggest that the fcd gene evolved in an unknown gene cluster involved in polysaccharide synthesis in some other bacteria and was transferred to A. actinomycetemcomitans.
6-Deoxy-D-talose, a component of serotype a-specific
antigen, and 6-deoxy-L-talose, a component of serotype
c-specific antigen, are also rare 6-deoxyhexoses (43). The
physiological significance of the rarity of 6-deoxyhexoses in the
serotype-specific antigens of A. actinomycetemcomitans
remains to be elucidated.
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FOOTNOTES |
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* This work was supported in part Grants-in-Aid for Scientific Research 10307054 and 11470452 from the Ministry of Education, Science, Sports and Culture, Tokyo, Japan and by a research grant from the Fund for Comprehensive Research on Aging and Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Preventive Dentistry, Kyushu University Faculty of Dentistry, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Tel.: 81-92-642-6423; Fax: 81-92-642-6354; E-mail: yosh{at}dent.kyushu-u.ac.jp.
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ABBREVIATIONS |
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The abbreviations used are: dTDP, thymidine diphosphate; PAGE, polyacrylamide gel electrophoresis; dTTP, thymidine triphosphate; HPLC, high performance liquid chromatography; ORF, open reading frame; PCR, polymerase chain reaction.
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