Guanosine diphosphate-4-keto-6-deoxy- D -mannose reductaseNao Suzuki1, Yoshio Nakano1, Yasuo Yoshida1, Takashi Nezu2, Yoshihiro Terada2, Yoshihisa Yamashita3 and Toshihiko Koga1 1 Depart
Trang 1Guanosine diphosphate-4-keto-6-deoxy- D -mannose reductase
Nao Suzuki1, Yoshio Nakano1, Yasuo Yoshida1, Takashi Nezu2, Yoshihiro Terada2, Yoshihisa Yamashita3 and Toshihiko Koga1
1
Department of Preventive Dentistry and2Department of Prosthetic Dentistry I, Kyushu University Faculty of Dental Science, Fukuoka, Japan;3Department of Oral Health, Nihon University School of Dentistry, Tokyo, Japan
The serotype a-specific polysaccharide antigen of
Actinoba-cillus actinomycetemcomitansis an unusual sugar,
6-deoxy-D-talose Guanosine diphosphate (GDP)-6-deoxy-D-talose is
the activated sugar nucleotide form of 6-deoxy-D-talose,
which has been identified as a constituent of only a few
microbial polysaccharides In this paper, we identify two
genes encoding GDP-6-deoxy-D-talose synthetic enzymes,
GDP-a-D-mannose 4,6-dehydratase and
GDP-4-keto-6-deoxy-D-mannose reductase, in the gene cluster required for
the biosynthesis of serotype a-specific polysaccharide
anti-gen from A actinomycetemcomitans SUNYaB 75 Both
gene products were produced and purified from Escherichia
colitransformed with plasmids containing these genes Their
enzymatic reactants were analysed by reversed-phase HPLC (RP-HPLC) The sugar nucleotide produced from GDP-a-D-mannose by these enzymes was purified by RP-HPLC and identified by electrospray ionization-MS,1H nuclear magnetic resonance, and GC/MS The results indicated that GDP-6-deoxy-D-talose is produced from GDP-a-D-mannose This paper is the first report on the GDP-6-deoxy-D-talose biosynthetic pathway and the role of GDP-4-keto-6-deoxy-D-mannose reductase in the synthesis
of GDP-6-deoxy-D-talose
Keywords: Actinobacillus actinomycetemcomitans; 6-deoxy-talose; NMR; polysaccharide; serotype-specific antigen
Capsular polysaccharides are ubiquitous structures found
on the cell surfaces of a broad range of bacterial species The
polysaccharides often constitute the outermost layer of the
cell, and have been implicated as an important factor in the
virulence of many animal and plant pathogens These
molecules are prominent structurally, and are serologically
diverse antigens that are involved in pathogenic processes
and in mediating resistance to host defense mechanisms [1]
Actinobacillus actinomycetemcomitans is a nonmotile,
Gram-negative, capnophilic, fermentative coccobacillus
that has been implicated in the aetiology and pathogenesis
of localized juvenile periodontitis [2–4], adult periodontitis
[5], and severe nonoral human infections [6] Traditionally,
A actinomycetemcomitans strains were divided into five
serotypes (a, b, c, d and e)[7–9], but recently a new serotype,
f, was reported [10] The serologic specificity is defined by
the polysaccharides on the surface of the organism [11] and the serotype-specific polysaccharide antigens (SPAs)are the immunodominant antigens in the organism [12–16] Serotype a-specific polysaccharide antigen from A actino-mycetemcomitans is a 6-deoxy-D-talan composed of repeating disaccharide units, which are acetylated at the O-2 position of 1,3-linked 6-deoxy-D-talose: )3))6-deoxy-a-D-talose-(1–2))6-deoxy-a-D-talose-(1– [17,18] Bacterial extracellular polysaccharides consisting solely of 6-deo-xytalose are rare Except for the serotype a-specific polysaccharide antigen from A actinomycetemcomitans, the exopolysaccharide isolated from Pseudomonas plantarii strain DSM 6535 is the only reported homopolysaccharide
of 6-deoxy-D-talose [19] The repeating unit of the exopolysaccharide from P plantarii has a different struc-ture: it is a trisaccharide that is acetylated at the O-2 position of 1,3-linked 6-deoxy-D-talose: )3))6-deoxy-a-D -talose-(1–2))6-deoxy-a-D-talose-(1–2)-6-deoxy-a-D -talose-(1– Other SPAs of A actinomycetemcomitans also contain rare sugars as constituents of microbial polysaccharides; examples include D-fucose in serotype b-specific polysac-charide antigen [12] and 6-deoxy-L-talose in serotype c-specific polysaccharide antigen [17]
The mechanism for the biosynthesis of GDP-6-deoxy-D -talose, which is the activated sugar nucleotide form of 6-deoxy-D-talose, is unknown It is thought that GDP-6-deoxy-D-talose is formed from a-D-mannose-1-phosphate and GTP in three steps; the first two steps are common to the GDP-L-fucose, GDP-D-rhamnose, and GDP-L-colitose synthesis pathways, producing GDP-4-keto-6-deoxy-D -mannose (Fig 1)[20–22] a-D-Mannose-1-phosphate guanylyltransferase (ManC)combines a-
-mannose-1-Correspondence to Y Nakano, Department of Preventive Dentistry,
Kyushu University Faculty of Dental Science,
Fukuoka 812-8582, Japan.
Fax: + 81 92 642 6354, Tel.: + 81 92 642 6423,
E-mail: yosh@dent.kyushu-u.ac.jp
Abbreviations: ESI, electrospray ionisation; Gmd, GDP-a- D -mannose
4,6-dehydratase; Rmd, GDP-4-keto-6-deoxy- D -mannose reductase;
RP-HPLC, reversed-phase HPLC; SPA, serotype-specific
polysac-charide antigen.
Note: This work is dedicated in fondest memory to Prof T Koga,
whose influence as a mentor will be greatly missed and without whom
this work would not have been possible.
(Received 4 August 2002, revised 1 October 2002,
accepted 23 October 2002)
Trang 2phosphate with GTP to produce GDP-a-D-mannose Then,
GDP-a-D-mannose is converted into
GDP-4-keto-6-deoxy-D-mannose by GDP-a-D-mannose 4,6-dehydratase (Gmd)
GDP-D-rhamnose is then produced from
GDP-4-keto-6-deoxy-D-mannose by GDP-4-keto-6-deoxy-D-mannose
reductase (Rmd) GDP-6-deoxy-D-talose is a stereoisomer
of GDP-D-rhamnose at C4 GDP-6-deoxy-D-talose can be
synthesized by another GDP-4-keto-6-deoxy-D-mannose
reductase and the stereoselectivity of the reduction
deter-mines the direction of synthesis of these two
6-deoxyhex-oses However, neither the gene encoding the biosynthesis of
GDP-6-deoxy-D-talose nor its corresponding protein has
been found
Recently, we cloned and characterized a gene cluster
involved in the biosynthesis of SPA from A
actinomyce-temcomitansSUNYaB 75 (serotype a)(Fig 2A)[23] In a
protein database search the ORF9 product shared 52.0%
identity with the gmd gene product of Yersinia
pseudotu-berculosis[24] and the ORF7 product was 28.0% identical
with the rmd gene product of Pseudomonas aeruginosa [25]
We predicted that ORF9 and ORF7 encoded GDP-a-
-mannose 4,6-dehydratase and GDP-4-keto-6-deoxy-D -man-nose reductase in the biosynthesis of
GDP-6-deoxy-D-talose, respectively The gmd gene was subcloned into pIVEX2.3 and the tld gene was subcloned into pIVEX2.3MCS, and these gene products overproduced in Escherichia coliwere purified and characterized
Fig 2 Restriction map and genetic organization of the gene cluster responsible for the production of the SPA of A actinomycetemcomitans SUNYaB75 (serotype a) (A) and gel electrophoresis of recombinant enzymes purified from E coli strains transformed with the expression plasmids (B) (A)Closed arrows indicate ORFs The functions of the gene products predicted by homology search, the GC content of each ORF, and the SPA phenotypes caused by specific insertion mutants are shown in descending order below the restriction map A flag indicates the putative promoter The horizontal lines show the DNA fragments inserted into pMCL210 used for nucleotide sequencing Abbreviations: H, HindIII; E, EcoRI; A, Acc65I; Pa, PacI; Pm, PmeI; Tld, a putative GDP-4-keto-6-deoxy- D -mannose reductase; Ac-TRase, acetyltransferase; Gmd, GDP-a- D -mannose 4,6-dehydratase; XylR, xylose operon regulatory protein (B)Approximately 0.5 lg of each protein was incubated at 100 C in a water bath for 5 min with 0.1% (w/v)SDS and 1% (v/v)2-mercaptoethanol Each of the treated solutions was electrophoresed on a 12.5% SDS-polyacrylamide gel, which was stained with Coomassie Blue Lane 1, Purified gmd gene product (SUNYaB 75); lane 2, purified tld gene product; lane 3, purified gmd gene product (K12) The positions of molecular mass markers (kDa)are shown on the left.
Fig 1 Pathway for the synthesis of GDP- D -rhamnose, GDP- L -fucose,
and GDP-6-deoxy- D -talose from a-mannose-1-phosphate and GTP.
Asterisks above the parentheses indicate the genes encoding the
enzymes in A actinomycetemcomitans SUNYaB 75.
Trang 3E X P E R I M E N T A L P R O C E D U R E S
Bacterial strains, plasmids, and culture conditions
E coliDH5a (supE44 DlacU169 (/80 lacZDM15) hsdR17
recA1 endA1 gyrA96 thi-1 relA1)[26] was used for the DNA
manipulations and as the host strain for pIVEX2.3 and
pIVEX2.3MCS derivatives (Roche Molecular
Biochemi-cals) E coli ER2566 (F– k– fhuA2 (lon) ompT lacZ::T7
gene1 gal sulA11 D(mcrC-mrr)
114::IS10R(mcr-73::mini-Tn10-TetS)2 R(zgb-210::Tn10)(TetS) endA1 (dcm)) (New
England Biolabs)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 medium at 37 C Ampicillin was
used at a final concentration of 50 lgÆmL)1 The DNA
fragments carrying the gmd and tld genes of A
actinomyce-temcomitans SUNYaB 75 were amplified by PCR using
pSAA212 [23] as a template pSAA212 contains a 10.6-kb
Acc65I fragment responsible for the biosynthesis of serotype
a-specific polysaccharide antigen in A
actinomycetemcom-itansSUNYaB 75
DNA manipulation, PCR, and sequencing techniques
DNA fragment preparation, agarose gel electrophoresis,
DNA labelling, ligation, and bacterial transformation were
performed using the methods described by Sambrook et al
[26] PCR amplification was performed using T3
Thermo-cycler (Biometra, Go¨ttingen, Germany) Sequencing was
performed using an ABI 373A or an ABI PRISM 310
DNA sequencer (Applied Biosystems)
Construction of plasmid
Each DNA fragment carrying the gmd and tld genes of
A actinomycetemcomitansSUNYaB 75 was amplified by
PCR using pSAA212 [23] as a template To construct
plasmids for gene expression and protein purification, the
following sets of primers were designed to introduce
appropriate restriction sites for subcloning: to subclone
the gmd gene into the vector pIVEX2.3, 5¢-CGCG
CCATGGTGAAAACAGCAATTGTAACT-3¢ (NcoI)
and 5¢-GCGCCCCGGGAAAAGAAAAACC-3¢ (SmaI);
and to subclone the tld gene into the vector pIVEX2.3MCS,
5¢-GCGCCATATGAAAATCTTAGTA-3¢ (NdeI)and
5¢-GCGCCCCGGGAATCGAAAGCTC-3¢ (SmaI) Each
PCR product was purified using a QIAquick PCR
Purifi-cation Kit (QIAGEN GmbH)and, after double digestion
with the appropriate restriction enzymes, directly ligated
into the vector plasmid, which had been cleaved with the
same enzymes Plasmids containing the gmd and tld genes
bound to a His6-tag were constructed using the vectors
pIVEX2.3 and pIVEX2.3MCS, respectively The DNA
fragment carrying the gmd gene of E coli K12 was
amplified by PCR using chromosomal DNA of E coli
DH5a as a template with the following primers:
5¢-CGCGCATATGTCAAAAGTCGCTCTCATC-3¢ (NdeI)
and 5¢-ATATCCCGGGTGACTCCAGCGCGATCGC-3¢
(SmaI) After purification and double digestion with NdeI
and SmaI, the fragment was directly ligated into NdeI–SmaI
double-digested pTYB2 vector (New England Biolabs)
Enzyme purification
To purify the gmd and tld proteins bound to the His6 -tag, E coli DH5a harbouring the expression plasmids was grown in 50 mL 2· TY cultures supplemented with
50 lgÆmL)1 ampicillin at 37C for 16 h After the cells had been harvested and disrupted by ultrasonication (Heat Systems-Ultrasonics Inc., Plainview, USA), cell extracts were obtained by centrifugation at 20 000 g for
20 min at 4C Purification was based on affinity chromatography using chelate-absorbent nickel–nitrilotri-acetic acid resin (Qiagen), which interacted with the His6 -tag To purify the gmd product in E coli K12, E coli ER2566 transformed pTYB2 containing the gmd gene was grown in 500 mL 2· TY broth with ampicillin at
37C to an optical density of 0.7 at 600 nm The culture was induced with 1 mM isopropyl-b-thiogalactopyrano-side The cells were harvested 4 h after induction and lysed by ultrasonication The cell extract was obtained by centrifugation at 20 000 g for 30 min at 4C Binding
of the fusion proteins to chitin beads via the intein/ chitin binding domain, cleavage of the fusion protein (in
20 mMTris/HCl, pH 8.0, 200 mM NaCl, 0.1 mM EDTA,
30 mM dithiothreitol at 4C), and elution of product were all carried out according to the manufacturer’s instructions
Enzyme assay The conversion of GDP-a-D-mannose to GDP-4-keto-6-deoxy-D-mannose or GDP-6-deoxy-D-talose was detected
by reversed-phase HPLC (RP-HPLC)(Waters, Milford, USA) The standard mixture contained 50 mM sodium phosphate buffer, pH 7.2, 12 mMMgCl2, 8 mMGDP-a-D -mannose, 12 mMNADPH, and 2 lg of the purified gene products per mL The reactions were performed in the same
one-pot assay and incubated at 37C for 3 h
Detection and purification of sugar nucleotides
by RP-HPLC Sugar nucleotides in the reaction mixtures of the gene products were identified using RP-HPLC as described by Albermann et al [27] Samples (10 lL)diluted 10-fold with distilled water were injected onto a TSKgel ODS-80Ts column (0.46· 15 cm; Tosoh, Tokyo, Japan)with a phosphate buffer [30 mM potassium phosphate, pH 6.0,
5 mM tetrabutylammonium hydrogen sulfate, 2% (v/v) acetonitrile] as the mobile phase at a flow rate of 1.0 mLÆmin)1at 40C The eluate was monitored with a
UV detector at 254 nm
The predictive GDP-6-deoxy-D-talose was pooled from repeated RP-HPLC runs on the ODS-80Ts column, as described by Tonetti et al [28] For collection 0.5M
KH2PO4 was used as the mobile phase to cut down the running time The fraction from each run was immediately cooled on ice to prevent degradation in 0.5MKH2PO4at room temperature After removing the excess phosphate by adding 4 vols cold 100% ethanol, the solution was freeze-dried using a DC41 freeze dryer (Yamato, Tokyo, Japan) and lyophilized The purified sugar nucleotide was stored at )30 C
Trang 4Electrospray ionization-MS (ESI/MS)
To remove completely the phosphate and replace the
solvent, RP-HPLC (HP 1100 Series; Hewlett-Packard)was
used The predictive GDP-6-deoxy-D-talose was
chroma-tographed on a TSKgel Super-ODS column (0.46· 5 cm;
Tosoh)with 0.1% formic acid as the mobile phase at a
flow rate of 0.2 mLÆmin)1 The eluate was monitored with
a UV detector at 254 nm The collected fractions were
used for ESI/MS with a Mariner Biospectrometry
Work-station (Perkin-Elmer, Norwalk, USA) The mass was
scanned from m/z 500–700 at a 90-V nozzle potential in
the positive ion mode by manual injection at a rate of
5.0 lLÆmin)1
1H NMR spectroscopy
Approximately 2-mg samples were dissolved in D2O and
freeze-dried again to remove any H2O completely; then
each sample in 0.5 mL of D2O was transferred to a 5-mm
NMR tube 1H NMR spectra were recorded with a
Bruker AM400 spectrometer (Rheistetten, Germany) The
measurements were made at 298 K The chemical shifts
were referenced to 3-(trimethylsilyl)propanesulfonic acid at
0.0 p.p.m The1H spectra of 64 scans were recorded with
presaturation of the HOD resonance at 4.72 p.p.m
Two-dimensional COSY measurement was also performed for
signal assignments
GC/MS
The predicted GDP-6-deoxy-D-talose was obtained by the
method described in Detection and purification of sugar
nucleotides by RP-HPLC The glycoside of serotype
a-specific polysaccharide antigen, which consists of only
6-deoxy-D-talose, was purified from an autoclaved extract
of A actinomycetemcomitans ATCC 29523 by the
method of Amano et al [12] The glycoside of serotype
c-specific polysaccharide antigen, which consists of only
6-deoxy-L-talose, was extracted from A
actinomycetem-comitans NCTC 9710 by the method of Yoshida et al
[29]
Samples of 2 mg were dissolved in 200 lL 0.1MHCl
The ampoules containing the solutions were sealed under
vacuum and heated at 80C for 1 h to hydrolyse them; they
were then dried to remove the water and HCl The pellets
were converted into the correspondingD
-(+)-2-octylglyco-side acetate by the method of Leontein et al [30] The sugar,
one drop of trifluoroacetic acid, and D-(+)-2-octanol
(300 lL)were transferred to an ampoule After sealing the
ampoule and heating it at 130C for 16 h, the ampoule
contents were evaporated at 55C Each product was kept
at 100C for 20 min in acetic anhydride-pyridine (1 : 1,
50 lL), and characterized by TurboMass GLC/MS
(Per-kin-Elmer)using a fused silica capillary column (CP Sil-88,
0.25 mm· 50 m; Chrompack Inc., Bridgewater, NJ, USA)
at 200C Approximately 5 lL of sample were injected, and
the split ratio was 1 : 20 Helium was used as the carrier gas
at a flow rate of 0.9 mLÆmin)1 Ionization was performed by
electron impact The fragment ionization peaks were
analysed under an ionization potential of 70 eV A library
search of mass chromatograms was performed using NIST
Search
R E S U L T S Purifying the enzymes involved in the synthesis
of GDP-6-deoxy-D-talose
To characterize the function of the gmd and tld gene products in A actinomycetemcomitans SUNYaB 75, the gene products were purified by affinity chromatography as described in detail in Experimental procedures The molecular masses of the denatured polypeptides, determined
by SDS/PAGE to be 38.9, 33.4, and 42.0 kDa agree with the predicted His6-tagged gmd (SUNYaB 75), His6-tagged tld, and gmd (K12)gene products, respectively (Fig 2B) The His6-tagged gmd and tld gene products were not completely homogeneous, as judged by SDS/PAGE To determine unequivocally the His6-tagged proteins, Western blotting was performed with RGS–His antibody (Qiagen) Single bands of the expectative sizes were observed speci-fically in both (data not shown)
Identifying GDP-6-deoxy-D-talose from GDP-a-D-mannose by RP-HPLC and ESI/MS analysis Conversion of GDP-a-D-mannose into GDP-sugars was detected by RP-HPLC (Fig 3) The elution profile of the reaction mixture containing GDP-a-D-mannose, NADPH, and the gmd gene product homologue from A actinomyce-temcomitansSUNYaB 75 (Fig 3B)or E coli K12 (Fig 3C) are shown GDP-4-keto-6-deoxy-D-mannose was detected
as a broad peak (42.0 min) Both reactions involving the gmd gene products halted after consuming some of the GDP-a-D-mannose, regardless of the addition of the proteins The peak that appeared at 24.0 min was in agreement with that of authentic NADP+ The reason why the NADP+ peak appeared in the gmd gene product reaction has been unidentified The retention time of the putative GDP-6-deoxy-D-talose was 36.0 min in the reac-tion mixture containing GDP-a-D-mannose, NADPH, and the gmd and tld gene products of A actinomycetemcomitans SUNYaB 75 (Fig 3D) To determine the mass of this final product, it was purified and analysed by ESI/MS (Fig 4) The peak in the ESI/MS spectrum of the product was at 590.1, which corresponds to the [M + H]+ion of GDP-6-deoxy-D-talose
1H NMR analysis of the structure of the purified GDP-6-deoxyhexose
Approximately 2 mg of the sugar nucleotide obtained from the enzyme assay using the gmd and tld gene products were pooled from several RP-HPLC runs on the ODS-80Ts After removing the excess phosphate by adding ethanol, the solution was concentrated by freeze-drying The concentrated solution was lyophilized and dissolved in D2O The NMR spectra of authentic GTP and GDP-a-D-mannose were also measured The spectra
of authentic GTP and GDP-a-D-mannose, and the sugar nucleotide are shown in Fig 5 Assignment of these resonances was verified in two-dimensional homonuclear
1H-1H COSY experiments (data not shown) The assigned chemical shifts and coupling constants are summarized in Table 1 The signals for the nucleotide moieties in the GDP-sugars were in good agreement with those of GDP
Trang 5The signals for H2¢ of the nucleotide moieties overlapped
that of HOD, and the H5¢¢ and H6¢¢ signals of the sugar
moieties of GDP-a-D-mannose also overlapped The
signals for the sugar moiety of the predicted
GDP-6-deoxy-D-talose were H6¢¢ (1.18 p.p.m., doublet), H4¢¢
(3.64 p.p.m., double doublet), H5¢¢ (3.88 p.p.m.,
multi-plet), H3¢¢ (3.91 p.p.m., double doublet), H2¢¢ (4.03 p.p.m.,
double doublet)and H1¢¢ (5.50 p.p.m., doublet) From the
observed coupling constants J(1,2)¼ 5.40 Hz,
J(2,3)¼ 3.40 Hz, J(3,4)¼ 6.36 Hz and J(4,5)¼ 1.96 Hz,
the orientations of H1¢¢, H2¢¢, H3¢¢, H4¢¢ and H5¢¢ are
estimated to be equatorial, equatorial, axial, equatorial
and axial, respectively This does not conflict with the
structure of GDP-6-deoxy-D-talose Moreover, the con-formation of –CH3was equatorial in this sugar nucleotide Since rhamnose has the corresponding coupling constants
of 1.5 (J(1,2)) , 3.5 (J(2,3)) , 9.5 (J(3,4)) , and 9.5 (J(4,5))Hz, which are totally different from the current ones [31], we can exclude the possibility that the product was GDP-D -rhamnose and can conclude that GDP-6-deoxy-D-talose was selectively synthesized In Table 1, the value of J(1,2) comes from H2¢¢, which did not agree with that from H1¢¢ The coupling constant J(1,2) must be identical, regardless
of whether it comes from H1¢¢ or H2¢¢ It is, however, possible that the neighbouring phosphate groups affect the coupling constant [32,33]
Determining the absolute configuration of the talosyl residue in the GDP-6-deoxytalose by GC/MS
Based on the coupling constants in the1H NMR spectra, we determined that the sugar nucleotide is GDP-6-deoxytalose However, the prediction that the absolute configuration of the talosyl residue in the GDP-6-deoxytalose isDwas not supported by direct evidence GC/MS was performed to prove the hypothesis The GDP-6-deoxytalose, the purified SPAs of A actinomycetemcomitans ATCC 29523 (serotype a)and NCTC 9710 (serotype c)were hydrolysed, and then the talosyl residues were detected asD-(+)-2-octylglycoside acetates Examination of the mass chromatogram library produced four fragment ion peaks from 6-deoxytalose for the talosyl residues of the GDP-6-deoxytalose and the SPA
of ATCC 29523 (Fig 6A and B) The four peaks were thought to be two pyranosides and two franosides The retention times (13.6, 23.5, 40.8, and 49.0 min)of the
Fig 3 RP-HPLC profiles during synthesis of GDP-6-deoxy- D -talose:
GDP-a- D -mannose (1), NADP (2), GDP-4-keto-6-deoxy- D -mannose
(3), and GDP-6-deoxy- D -talose (4) Samples were injected onto a
TSKgel ODS-80Ts column (A)No enzyme was added to the reaction
mixture (B)The purified His 6 -tagged gmd gene product of A
actino-mycetemcomitans SUNYaB 75 was added to the reaction mixture (C)
The purified gmd gene product of E coli K12 were added to the
reaction mixture (D)The purified His 6 -tagged gmd and tld gene
products were added to the reaction mixture.
Fig 4 The ESI/MS spectra for the authentic GDP-a- D -mannose (A) and the reaction product GDP-a- D -mannose, NADPH, and the gmd and tld gene products of A actinomycetemcomitans SUNYaB75 (B).
Trang 6GDP-6-deoxytalose agreed well with those (13.6, 24.0, 41.1,
and 49.3 min)of the SPA of ATCC 29523 (serotype a),
which is 6-deoxy-D-talan Conversely, the retention times
(13.1, 22.0, and 22.7 min)of the fragment ion peaks derived
from 6-deoxy-L-talose in the SPA of NCTC 9710 (serotype c)
did not agree with the other retention times (Fig 6C) Thus,
it was determined that the talose in GDP-6-deoxytalose had
theDabsolute configuration
D I S C U S S I O N Previously, we cloned and characterized the gene clusters responsible for the biosynthesis of SPAs of A actinomyce-temcomitansserotypes a, b, c, d, and e [23,29,34–36] The gene cluster associated with the synthesis of SPA in
A actinomycetemcomitansSUNYaB 75 (serotype a)con-tains 14 ORFs (Fig 2A) A protein database search was performed with the programsFASTA[37] andBLASTat the National Institute of Genetics, Mishima, Japan The products of 11 genes, ORF2–ORF12, were homologous
to bacterial gene products involved in the biosynthesis of extracellular polysaccharides Only the proteins encoded by ORF3 and ORF4, ABC transport proteins, showed high identities (64.0 and 73.0%, respectively)to the proteins enco-ded by ORFs in the clusters responsible for synthesizing the SPAs in other serotypes of A actinomycetemcomitans The biosynthetic pathway for GDP-6-deoxy- -talose,
Fig 5.1H NMR spectra of GTP (A) and GDP-a- D -mannose (B), and
the purified GDP-hexose converted from GDP-a- D -mannose by the gmd
and tld gene products (C) The inset shows an expansion of the H4¢–
H4¢¢ region.
Fig 6 Gas-liquid chromatograph spectra of the acetylated D -(+)-2-octyl glycosides obtained from the hydrolysate of the purified GDP-6-deoxytalose (A)Glycosides of the purified serotype a-specific poly-saccharide antigen (ATCC 29523) (B) Glycosides of the hydrolysate of the purified GDP-hexose converted from GDP-a- D -mannose by the gmd and tld gene products (C)Glycosides of the purified serotype c-specific polysaccharide antigen (NCTC 9710) Arrows indicate the fragment ion peaks from 6-deoxytalose for the talosyl residues.
Trang 7which is the activated nucleotide sugar form of 6-deoxy-D
-talose, is predicted to be quite different from the pathways
for the precursors of serotype b-, c-, d-, and e-specific
polysaccharide antigens [38] Insertional inactivation of
ORF2, 3 and ORF7 through ORF12 resulted in loss of
the ability of A actinomycetemcomitans SUNYaB 75 cells
to produce the polysaccharide In these genes the ORF2
product shared 58.0% identity with the manC gene
product in E coli [39] The manC gene product is a
a-mannose-1-phosphate guanylyltransferase, which
con-verts GTP and a-mannose-1-phosphate into
GDP-a-D-mannose The ORF9 product shared 52.0% identity
with the GDP-a-D-mannose 4,6-dehydratase of Y
pseu-dotuberculosis [24] In general, GDP-a-D-mannose
4,6-dehydratase is an important enzyme converting
GDP-a-D-mannose to GDP-4-keto-6-deoxy-D-mannose in
the pathway of GDP-L-fucose biosynthesis in many
bacteria, plants and mammals [40] The ORF7 product
had 28.0% homology to the rmd gene product of
P aeruginosa[25], which reduces GDP-4-keto-6-deoxy-D
-mannose to GDP-D-rhamnose [20] GDP-6-deoxy-D-talose
is a configrational isomer of GDP-D-rhamnose The rmd
gene product is a reductase that reduces the C4 position of
GDP-4-keto-6-deoxy-D-mannose to GDP-D-rhamnose,
and we postulated that ORF7 in A
actinomycetemcomi-tans SUNYaB 75 encodes another reductase producing
GDP-6-deoxy-D-talose from GDP-4-keto-6-deoxy-D
-man-nose, in spite of sharing low identity (28.0%) Several
consensus domains exist in the tld and rmd gene products
Among these, the structure YXXXK is an important
conserved structure within the short-chain dehydrogenase/
reductase family [41] In addition, both the tld and rmd
gene products contain an NAD-binding domain,
GXXGXXG, located near the N-terminus The tld gene
product can utilize either NADPH or NADH, although
NADPH is used efficiently (data not shown)
dTDP-4-keto-L-rhamnose reductase in the biosynthesis of
dTDP-6-deoxy-L-talose in A actinomycetemcomitans NCTC 9710
(serotype c)also preferred NADPH as a cofactor over
NADH [33] For NCTC 9710, the retention time of the
NADP+ peak overlapped that of the dTDP-6-deoxy-
-talose peak in RP-HPLC, and NADH was used as the coenzyme
The gmd and tld gene products in A actinomycetem-comitans SUNYaB 75 were obtained as His6-tagged proteins The enzymatic activities of the purified His6 -tagged gmd and tld gene products were determined by RP-HPLC analysis Previously, the gmd gene product with a His6-tag bound at its N terminus was found to be enzymatically inactive, perhaps because the multiple His-extender peptide affected its protein structure and altered the accessibility of the NADP+-binding site [27] Consid-ering this, we constructed plasmids with the His6-tag bound to the C terminus
We reported the pathways of dTDP-D-fucose (Y4)and dTDP-6-deoxy-L-talose (NCTC 9710)syntheses in
A actinomycetemcomitans, previously [32,33] Sugar nucleotides were detected and collected by RP-HPLC with 0.5MKH2PO4buffer as the mobile phase In this study, the retention time (5.1 min)of the GDP-6-deoxy-D-talose profile was close to that (5.0 min)of the GDP-4-keto-6-deoxy-D-mannose profile with 0.5M KH2PO4 buffer, in spite of the different shapes of the two peaks We could effectively collect the GDP-6-deoxy-D-talose quickly using 0.5M KH2PO4 buffer as the mobile phase By contrast, for detection, 30 mM potassium phosphate (pH 6.0) containing 5 mM tetrabutylammonium hydrogen sulfate and 2% acetonitrile was used as the mobile phase to definitely separate the products in the reaction mixture To confirm that the intermediate is GDP-4-keto-6-deoxy-D -mannose, the gmd gene product of E coli K12 was used The gmd gene of E coli has been characterized [39,40] The retention time of the product profile in the enzyme assay by the gmd gene product derived from A actino-mycetemcomitansSUNYaB 75 was obtained as broad peaks
at 42.0 min, which agree with those from E coli K12 (Fig 3B and C)
The conversion of GDP-a-D-mannose into GDP-4-keto-6-deoxy-D-mannose stopped when about 50% of the GDP-a-D-mannose was used up Addition of the protein was not effective in advancing the reaction Conversely,
in the one-pot assay almost complete conversion
Table 1 NMR spectroscopic identification of GTP, GDP-a- D -mannose, and GDP-a-6-deoxy- D -talose (s)Singlet, (d)doublet, (t)triplet, (dd)double doublet, (m)multiplet An asterisk indicates that the signal is broad and weakly coupling with H-5¢ ND, not determined.
Proton
Chemical shift
d (p.p.m.)
Chemical shift
d (p.p.m.)
Coupling constant
J (Hz)
Chemical shift
d (p.p.m.)
Coupling constant
J (Hz)
Trang 8occurred It is possible that feedback inhibition of the
GDP-a-D-mannose 4,6-dehydratase occurs via the
GDP-6-deoxy-D-talose pathway in A actinomycetemcomitans
SUNYaB 75 In the enzyme assay using the purified
His6-tagged gmd and tld gene products in two successive
steps, the GDP-4-keto-6-deoxy-D-mannose was
com-pletely converted into GDP-6-deoxy-D-talose, but no
new GDP-4-keto-6-deoxy-D-mannose was produced (data
not shown) It is considered that when the tld gene
product was added, the gmd gene product might have
become inactive However, further detailed analysis has
not been carried out
In 1973, 6-deoxy-L-talose was characterized as an unusual
sugar, and the instability of dTDP-6-deoxy-L-talose, which
is the activated sugar nucleotide form of 6-deoxy-L-talose,
was reported [42] Furthermore, we reported that
dTDP-6-deoxy-L-talose was degraded in mild alkaline conditions
[33] GDP-6-deoxy-D-talose was more sensitive to alkaline
conditions and heat than dTDP-6-deoxy-L-talose For
example, after GDP-6-deoxy-D-talose collected from
ODS-80Ts was evaporated at room temperature using the
same method as for dTDP-6-deoxy-L-talose, the peak for
this sugar nucleotide disappeared from the RP-HPLC
elution profile (data not shown) In this study, freeze-drying
was used to concentrate the samples
GDP-4-keto-6-deoxy-D-mannose was also unstable Kneidinger et al reported
that the product produced from GDP-a-D-mannose by the
gmdgene product in A thermoaerophilus was unstable and
decomposed to form GMP and GDP, as judged by anion
exchange HPLC analysis [20]
The GC contents of the genes essential for SPA
biosynthesis in A actinomycetemcomitans SUNYaB 75
are lower than the average GC content (47.8%)of the
genes flanking them The GC contents of ORF2, ORF7,
and ORF9 were 37.2, 30.5 and 35.4%, respectively
(Fig 2A) It has been reported that genes encoding basic
cellular functions in A actinomycetemcomitans have an
average GC content of 48.0% [43] The GC content of the
region essential for the biosynthesis of SPA in the other
serotype strains (b–e)of A actinomycetemcomitans is also
lower than the average GC content of these genes (48.0%)
[29,34–36] A lower GC content has been found in gene
clusters involved in the synthesis of various bacterial
polysaccharides [44–46] These findings suggest the
inter-specific transfer of these genes from other species with a low
GC content to A actinomycetemcomitans [47]
In conclusion, we identified
GDP-4-keto-6-deoxy-D-mannose reductase, which converts
GDP-4-keto-6-deoxy-D-mannose into GDP-6-deoxy-D-talose in
A actinomycetemcomitansSUNYaB 75 (serotype a), and
revealed the enzymatic process involved in
GDP-6-deoxy-D-talose synthesis
A C K N O W L E D G E M E N T S
This work was supported in part by a Grant-in-Aid for Encouragement
of Young Scientists 13771265 (Y N.), 14771185 (Y Yo.) and a
Grant-in-Aid for Developmental Scientific Research 12557186 (Y Ya.)from
the Ministry of Education, Culture, Sports, Science and Technology,
Tokyo, Japan, by a research grant from the Takeda Science
Foundation (Y N.), and by Research Fellowships from the Japan
Society for the Promotion of Science for Young Scientists 13010070
(N S.).
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