Guanosine diphosphate-4-keto-6-deoxy- D -mannose reductase in the pathway for the synthesis of GDP-6-deoxy- D -talose in Actinobacillus actinomycetemcomitans Nao Suzuki 1 , Yoshio Nakano 1 , Yasuo Yoshida 1 , Takashi Nezu 2 , Yoshihiro Terada 2 , Yoshihisa Yamashita 3 and Toshihiko Koga 1 1 Department of Preventive Dentistry and 2 Department of Prosthetic Dentistry I, Kyushu University Faculty of Dental Science, Fukuoka, Japan; 3 Department of Oral Health, Nihon University School of Dentistry, Tokyo, Japan The serotype a-specific polysaccharide antigen of Actinoba- cillus actinomycetemcomitans is 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 coli transformed 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, 1 H 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- D -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) Eur. J. Biochem. 269, 5963–5971 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03331.x phosphate 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- temcomitans SUNYaB 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- D - 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 coli were purified and characterized. Fig. 2. Restriction map and genetic organization of the gene cluster responsible for the production of the SPA of A. actinomycetemcomitans SUNYaB 75 (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 lgofeach 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. 5964 N. Suzuki et al. (Eur. J. Biochem. 269) Ó FEBS 2002 EXPERIMENTAL PROCEDURES Bacterial strains, plasmids, and culture conditions E. coli DH5a (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)2R(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- itans SUNYaB 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. actinomycetemcomitans SUNYaB 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); andtosubclonethetld 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 His 6 -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 His 6 - tag, E. coli DH5a harbouring the expression plasmids was grown in 50 mL 2 · TY cultures supplemented with 50 lgÆmL )1 ampicillin at 37 °C 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 4 °C. Purification was based on affinity chromatography using chelate-absorbent nickel–nitrilotri- acetic acid resin (Qiagen), which interacted with the His 6 - 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 37 °C to an optical density of 0.7 at 600 nm. The culture was induced with 1 m M 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 4 °C. Binding of the fusion proteins to chitin beads via the intein/ chitin binding domain, cleavage of the fusion protein (in 20 m M Tris/HCl, pH 8.0, 200 m M NaCl, 0.1 m M EDTA, 30 m M dithiothreitol at 4 °C), 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 m M sodium phosphate buffer, pH 7.2, 12 m M MgCl 2 ,8m M GDP-a- D - mannose, 12 m M NADPH, and 2 lg of the purified gene products per mL. The reactions were performed in the same Ôone-potÕ assay and incubated at 37 °Cfor3h. 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 m M potassium phosphate, pH 6.0, 5m M tetrabutylammonium hydrogen sulfate, 2% (v/v) acetonitrile] as the mobile phase at a flow rate of 1.0 mLÆmin )1 at 40 °C.Theeluatewasmonitoredwitha UV detector at 254 nm. ThepredictiveGDP-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 0.5 M KH 2 PO 4 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 0.5 M KH 2 PO 4 at 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. Ó FEBS 2002 GDP-6-deoxy- D -talose synthetic enzyme (Eur. J. Biochem. 269) 5965 Electrospray 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 · 5cm; 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 . 1 H NMR spectroscopy Approximately 2-mg samples were dissolved in D 2 Oand freeze-dried again to remove any H 2 O completely; then each sample in 0.5 mL of D 2 O was transferred to a 5-mm NMR tube. 1 H 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. The 1 H 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 ÔDetection and purification of sugar nucleotides by RP-HPLCÕ. 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 lL0.1 M HCl. The ampoules containing the solutions were sealed under vacuumandheatedat80 °C for 1 h to hydrolyse them; they were then dried to remove the water and HCl. The pellets were converted into the corresponding D -(+)-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 130 °C for 16 h, the ampoule contents were evaporated at 55 °C. Each product was kept at 100 °C 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 200 °C. 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. RESULTS 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 His 6 -tagged gmd (SUNYaB 75), His 6 -tagged tld,andgmd (K12) gene products, respectively (Fig. 2B). The His 6 -tagged gmd and tld gene products were not completely homogeneous, as judged by SDS/PAGE. To determine unequivocally the His 6 -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- temcomitans SUNYaB 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. 1 H 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 D 2 O. 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 1 H- 1 H 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. 5966 N. Suzuki et al. (Eur. J. Biochem. 269) Ó FEBS 2002 The 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 –CH 3 was 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) ),and9.5(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 the 1 H 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 is D was 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 as D -(+)-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. actinom ycetem comitans SUNYaB 75 (B). Ó FEBS 2002 GDP-6-deoxy- D -talose synthetic enzyme (Eur. J. Biochem. 269) 5967 GDP-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 the D absolute configuration. DISCUSSION Previously, we cloned and characterized the gene clusters responsible for the biosynthesis of SPAs of A. actinomyce- temcomitans serotypes a, b, c, d, and e [23,29,34–36]. The gene cluster associated with the synthesis of SPA in A. actinomycetemcomitans SUNYaB 75 (serotype a) con- tains 14 ORFs (Fig. 2A). A protein database search was performed with the programs FASTA [37] and BLAST at 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- D -talose, Fig. 5. 1 H 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. 5968 N. Suzuki et al. (Eur. J. Biochem. 269) Ó FEBS 2002 which 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- L - 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 His 6 -tagged proteins. The enzymatic activities of the purified His 6 - tagged gmd and tld gene products were determined by RP- HPLC analysis. Previously, the gmd gene product with a His 6 -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 His 6 -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 0.5 M KH 2 PO 4 buffer 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 0.5 M KH 2 PO 4 buffer, in spite of the different shapes of the two peaks. We could effectively collect the GDP-6-deoxy- D -talose quickly using 0.5 M KH 2 PO 4 buffer as the mobile phase. By contrast, for detection, 30 m M potassium phosphate (pH 6.0) containing 5 m M 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- mycetemcomitans SUNYaB 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 Ôone-potÕ 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. GTP GDP-a- D -mannose GDP-a-6-deoxy- D -talose 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) H-8 8.10 (s) 8.09 (s) 8.09 (s) H-1¢ 5.91 (d) 5.92 (d) 5.91 (d) H-2¢ ND ND ND H-3¢ 4.55 (m) 4.50 (dd) 4.48 (dd) H-4¢ 4.34 (d*) 4.33 (d*) 4.32 (dd*) H-5¢ 4.22 (m) 4.19 (t) 4.16 (m) H-1¢¢ 5.50 (d) J 1,2 6.36 5.50 (d) J 1,2 5.40 H-2¢¢ 4.03 (d) J 2,3 2.92 4.03 (dd) J 2,3 3.40 H-3¢¢ 3.90 (dd) J 3,4 9.76 3.91 (dd) J 3,4 6.36 H-4¢¢ 3.66 (t) J 4,5 9.76 3.64 (dd) J 4,5 1.96 H-5¢¢ ND 3.88 (m) H-6¢¢ ND 1.18 (d) Ó FEBS 2002 GDP-6-deoxy- D -talose synthetic enzyme (Eur. J. Biochem. 269) 5969 occurred. 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 His 6 -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 gmd gene 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. actinomycetemcomitans SUNYaB 75 (serotype a), and revealed the enzymatic process involved in GDP-6-deoxy- D -talose synthesis. ACKNOWLEDGEMENTS 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. 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Biochem. 269) 5971 . by ORFs in the clusters responsible for synthesizing the SPAs in other serotypes of A. actinomycetemcomitans. The biosynthetic pathway for GDP-6-deoxy- D -talose, Fig Guanosine diphosphate-4-keto-6-deoxy- D -mannose reductase in the pathway for the synthesis of GDP-6-deoxy- D -talose in Actinobacillus actinomycetemcomitans Nao