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Glycobiology vol. 11 no. 8 pp. 655–661, 2001 Expression and identification of the RfbE protein from Vibrio cholerae O1 and its use for the enzymatic synthesis of GDP-D-perosamine Christoph Albermann and Wolfgang Piepersberg1 Chemische Mikrobiologie, Bergische Universität GH Wuppertal, Gauss-Str. 20, D-42097, Germany Received on January 15, 2001; revised on March 26, 2001; accepted on March 28, 2001 The 4-amino-6-deoxy-monosaccharide D-perosamine is an important element in the glycosylation of interesting cell products, such as antibiotics and lipopolysaccharides (LPS) of Gram-positive and Gram-negative bacteria. The biosynthetic pathway of the precursor molecule, GDP-D-perosamine, in Vibrio cholerae O1 starts with an isomerisation of fructose6-phosphate catalyzed by the bifunctional enzyme phosphomannose isomerase–guanosine diphosphomannose pyrophosphorylase (RfbA; E.C. 2.7.7.22) creating the intermediate mannose-6-phosphate, which is subsequently converted by the phosphomanno-mutase (RfbB; E.C. 5.4.2.8) and further by RfbA to GDP-D-mannose, to GDP-4 keto-6-deoxymannose by a 4,6-dehydratase (RfbD; E.C. 4.2.1.47) and finally to GDP-D-perosamine by an aminotransferase (RfbE; E.C. not yet classified). We cloned the rfbD and the rfbE genes of V. cholerae O1 in Escherichia coli expression vectors. Both biosynthetic enzymes were overproduced in E. coli BL21 (DE3) and their activities were analyzed. The enzymatic conversion from GDP-D-mannose to GDP-D-perosamine was optimized and the final product, GDP-D-perosamine, was purified and identified by nuclear magnetic resonance, mass spectrometry, and chromatography. The catalytically active form of the GDP-4-keto-6-deoxy-D-mannose-4-aminotransferase seems to be a tetramer of 170 kDa. The His-tag RfbE fusion protein has a Km of 0.06 mM and a Vmax value of 38 nkat/mg protein for the substrate GDP-4-keto-6deoxy-D-mannose. The Km and Vmax values for the cosubstrate L-glutamate were 0.1 mM and 42 nkat/mg protein, respectively. The intention of this work is to establish a basis for both the in vitro production of GDP-D-perosamine and for an in vivo perosaminylation system in a suitable bacterial host, preferably E. coli. Key words: GDP-α-D-perosamine/6-deoxyhexose/enzymatic synthesis/GDP-4-keto-6-deoxy-D-mannose 4-aminotransferase Introduction The 6-deoxyhexose D-perosamine is an important and characteristic element in glycoconjugates of bacteria. In Gram-positive 1To whom correspondence should be addressed © 2001 Oxford University Press bacteria, for example, the D-perosamine was first detected in the antibiotic perimycin of Streptomyces coelicolor var. aminophilus (Lee and Schaffner, 1966, 1969). In Gram-negative bacteria it is also found as a main constituent of lipopolysaccharides (LPS) in Brucella spp., Eschericha coli O157, and Vibrio cholerae O1 (Wu and Mackenzie, 1987; Bilge et al., 1996; Villeneuve et al., 2000). The O-antigen of V. cholerae O1, the causative agent of cholera, is a homopolymer of the dideoxy amino sugar perosamine, which is substituted via the amino group with a 4-carbon aliphatic acid, 3-deoxy-L-glycero-tetronic acid. This structure is repeated on the average 18 times and is joined to a lipid A molecule by a linker containing 6-, 7-, and 8-carbon sugars (core unit) (Hisatsune et al., 1993). The introduction of the perosamine monosaccharidic unit into the glycosylated endproducts is dependent on nucleotide activation as a GDPhexose and subsequent modification. The rfb region of V. cholerae O1 encodes the gene cluster that is responsible for the perosamine biosynthesis, the 3-deoxy-L-glycero-tetronate biosynthesis, the O-antigen transport, and of the O-antigen modification (Hisatsune et al., 1996; Stroeher et al., 1998). The perosamine biosynthesis cluster consists of four genes that are predicted to encode the enzymes for the conversion of fructose-6-phosphate to the activated sugar metabolite GDP-Dperosamine in five steps (Stroeher et al., 1995). The first step is the conversion of fructose-6-phosphate to mannose-6-phosphate catalyzed by the phosphomannose isomerase–guanosine diphosphomannose pyrophosphorylase (RfbA; E.C. 2.7.7.22); subsequently, the mannose-6-phosphate is converted to mannose-1-phosphate by the phosphomanno-mutase (RfbB; E.C. 5.4.2.8). The third step, the nucleotide activation, is catalyzed once again by the bifunctional phosphomannose isomerase–guanosine diphosphomannose pyrophosphorylase (RfbA) to GDP-D-mannose. The two further biosynthetic steps characteristic for the formation GDP-D-perosamine basically follow that of other 6-deoxyhexoses, which are made via the dTDP- or CDP-activated sugars. It starts with a dehydratation reaction catalyszed by an NAD+-dependent NDP-hexose 4,6dehydratase, which forms NDP-6-deoxy-D-4-hexulose (Piepersberg, 1994; Liu and Thorson, 1994; Piepersberg and Distler, 1997). By further reactions this intermediate can be modified by a large variety of following steps, for example, the 4-carbonyl group can be enantioselectively reduced, transaminated, or fully reduced. In other cases the NDP-6-deoxy-D-4hexuloses are converted first by an 3,5-epimerase reaction to NDP-6-deoxy-L-4-hexulose. In the case of GDP-D-perosamine, it is supposed that the activated sugar metabolite GDP-Dmannose is converted by the GDP-D-mannose 4,6-dehydratase (RfbD; E.C. 4.2.1.47) to GDP-4-keto-6-deoxy-D-mannose 655 C. Albermann and W. Piepersberg followed by a transamination catalyzed by the perosamine synthetase (RfbE; E.C. not yet classified) (Figure 1). To produce novel glycoconjugates or oligosaccharides by the use of glycosyltransferases, it is necessary to synthesize the required sugar donors in a large scale. Other nucleotideactivated sugars, for instance, GDP-D-mannose, GDP-Lfucose, or dTDP-L-rhamnose, are already available fully on the basis of enzymatic reactions (Elling et al., 1996; Pastuszak et al., 1998; Elling and Bütler, 1999; Albermann et al., 2000) with the respective enzymes overproduced in a suitable expression system such as E. coli. In this article we describe the cloning and the overproduction of the RfbE (GDP-D-perosamine synthetase) from V. cholerae O1. This work presents the evidence that the final product of the enzymatic reaction catalyzed by RfbE-protein is GDP-α-D-perosamine. Results Cloning and expression of GDP-D-mannose 4,6-dehydratase (RfbD) and GDP-D-perosamine synthetase (RfbE) analysis. Additionally the genes were cloned into the pET16b expression vector to obtain their products as N-terminal His-tag fusion proteins. The plasmids pCAW 13.1 (rfbD), pCAW 13.2 (His-tag rfbD), pCAW 14.1 (rfbE), and pCAW 14.2 (His-tag rfbE) constructed this way were transformed into E. coli BL21 (DE3) pLysS, where their expression under induced conditions resulted in the overproduction of large amounts of RfbD and RfbE proteins. The apparant molecular masses in analyses by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) agreed with the calculated masses of the proteins (RfbD 42.0 kDa; His-tag RfbD 44.5 kDa; RfbE 40.9 kDa; His-tag RfbE 43.4 kDa). Crude ribosome-free extracts from the recombinant E. coli strains were preliminarily tested for the activities of GDP-D-mannose 4,6-dehydratase and of GDP-4keto-6-deoxy-D-mannose 4-aminotransferase (GDP-D-perosamine synthetase). The recombinant native RfbD protein and the His-tag fusion protein variant of RfbD did not show any activity. Therefore the recombinant GDP-D-mannose 4,6dehydratase (Gmd; plasmid pCAW21.1) of E. coli K12 was The two open reading frames rfbD and rfbE of the LPS biosynthesis cluster of V. cholerae strain O1 (Stroeher et al., 1992; Manning et al., 1995) are predicted to encode a GDP-Dmannose 4,6-dehydratase and GDP-D-perosamine synthetase. The RfbD protein has high similarity with other 4,6-dehydratases from E. coli, Salmonella enterica, Yersinia enterocolitica, or Helicobacter pylori. The RfbE protein possesses a pyridoxal-phosphate-binding domain, which is supposed to be a group VI (secondary metabolic) aminotransferase and is in the cluster of orthologous proteins COG0399 (Tatusov et al., 2001) (Figure 2). Both genes were amplified and modified by polymerase chain reaction (PCR), respectively, to introduce each an N-terminal NdeI, overlapping the ATG start codon, and a BamHI restriction site downstream from the stop codon. These were used to clone the respective PCR products into the expression vector pET11a and were verified by sequence Fig. 1. Biosynthetic pathway of GDP-α-D-perosamine. The steps are catalysed by the following enzymes: (1) Phosphomannose isomerase–guanosine diphosphomannose pyrophosphorylase (RfbA), (2) phosphomannomutase (RfbB), (3) phosphomannose isomerase–guanosine diphosphomannose pyrophosphorylase (RfbA), (4) GDP-α-D-mannose 4,6-dehydratase (RfbD, Gmd), (5) GDP-4-keto-6-deoxy-α-D-mannose 3,5-epimerase-4-reductase (RfbE). 656 Fig. 2. Alignment of the V. cholerae O1 RfbE protein (acc. no. X59554 ) (1) with amino acid sequences of (2) the GDP-perosamine-synthetase PerA from Brucella melitensis 16M (acc. no. AF047478), (3) LmbS from Streptomyces lincolnensis 78–11 (acc. no. X79146) and (4) StrS from Streptomyces griseus N2311 (acc. no. Y00459). All four proteins are in cluster COG0339 (Tatusov et al., 2001). RfbE protein from V. cholerae used; it had previously already been shown to catalyze the synthesis of GDP-4-keto-6-deoxy-D-mannose (Sturla et al., 1997; Albermann et al., 2000). This finding was surprising, because the amino acid sequence of the RfbD protein (V. cholerae O1) and the Gmd protein (E. coli K12) have 79% identity and 90% homology. In contrast, both the native RfbE protein and its His-tag fusion variant were catalytically active in the conversion of GDP-4-keto-6-deoxy-D-mannose to GDP-D-perosamine. Purification and properties of the His-tag fusion protein RfbE After harvesting of E. coli BL21 (DE3) pLysS cells containing the plasmid pCAW14.2, a crude extract was prepared and chromatographed on a Ni-NTA-agarose column, which bound the His-tag RfbE fusion protein tightly. The elution of the recombinant protein from the affinity column was accomplished by increasing the imidazole concentration up to 250 mM. The imidazole was removed from the protein fraction by gel filtration. The eluted fractions each contained one major band in an SDS–PAGE analysis (Figure 3) consistent with the calculated molecular masses of His-tag Gmd (44.5 kDa) and of His-tag RfbE (43.4 kDa), respectively. A gel permeation analysis was used to investigate the molecular mass of the His-tag RfbE fusion protein under native conditions. The apparent mass of 170 kDa, measured by comparison of the elution volume of the His-tag RfbE peak with those of known molecular weight standards, suggested that the native protein is a tetramer. Determination of enzyme specificity and specific activity The conversion of GDP-α-D-mannose to GDP-4-keto-6-deoxyamnnose and futher to GDP-D-perosamine was monitored by high-performance liquid chromatography (HPLC) (Figure 4). In coupled reaction assays, with recombinant Gmd and RfbE or His-tag RfbE, the complete conversion of GDP-α-Dmannose to GDP-D-perosamine could be obtained. As an amino-donor out of the tested alternatives the amino acid Lglutamate was used as substrate of the RfbE protein. From the other tested aminodonors (L-alanine, L-arginine, L-aspartate, L-glycine, L-glutamine, L-leucine, L-lysine, L-ornithine, L-phenylalanine, L-serine) only L-glutamine showed activity, for which the conversion rate was only 10% of the activity compared with L-glutamate. It was also shown that the addition of the cosubstrate pyridoxal-5-phosphate was essential for catalysis of the aminotransfer reaction by RfbE. The specific activities of the crude extract of E. coli BL21(DE3) pLysS pCAW 14.2 and of the purified His-tag fusion protein under the given reaction conditions were 8 and 44 nkat/mg protein, respectively. This indicated approximately a 6.5-fold enrichment of the purified protein. In the negative controls, that is, crude extracts from E. coli BL21 (DE3) pLysS with either vector plasmid, pET11a, or pET16b, no activity of either GDP-D-mannose 4,6dehydratase or GDP-D-perosamine synthetase could be detected under the same conditions. For the determination of the kinetic properties the concentration of GDP-4-keto-6-deoxy- D-mannose was measured by the UV absorption in alkaline medium at 320 nm. The Km and Vmax values for the substrate GDP-4-keto-6-deoxy-D-mannose were 0.06 mM and 38 nkat/mg protein, respectively. The Km and Vmax values for the cosubstrate L-glutamate were 0.1 mM and 42 nkat/mg protein, respectively. The reaction catalyzed by the His-tag RfbE protein had its pH optimum at 8.0, and the optimum concentration of the divalent cation Mg2+ was 20 mM. Preparative synthesis and isolation of GDP-α-D-perosamine To identify the product of the RfbE reaction, we used the overproduced enzyme GDP-D-mannose 4,6-dehydratase (Gmd) and the purified GDP-D-perosamine synthetase (His-tag RfbE) for a preparative conversion of GDP-α-D-mannose. No product inhibition of the GDP-D-mannose 4,6-dehydratase by Fig. 3. Purification of the His-tag RfbE protein by Ni-NTA-agarose. The SDS–PAGE analysis of the following protein fractions is shown in the lanes: (1) Low molecular weight marker; (2) S30 of E. coli BL21 pLysS pCAW14.2 (His tag RfbE); (3) wash step 1 with 20 mM imidazole; (4) wash step 2 with 50 mM imidazole; (5) His-tag RfbE after purification on Ni-NTA-agarose and gel filtration; (6) elution step with 250 mM imidazole; (7) S30 of E. coli BL21 pLysS pCAW14.1 (native RfbE). Fig. 4. HPLC analysis of the enzymatic reactions catalyzed by Gmd and RfbE. (A) Enzyme assay after 1 min of incubation, GDP-α-D-perosamine (7 min), GDP-α-D-mannose (24 min), GDP-4-keto-6-deoxy-D-mannose (37 min). (B) Enzyme assay after complete conversion, GDP-α-D-perosamine (7 min). 657 C. Albermann and W. Piepersberg GDP-D-perosamine could be observed. Such a one-pot method was applied to the complete synthesis of GDP-D-perosamine. The synthesis was started from GDP-α-D-mannose and the formation of the product GDP-D-perosamine and the consumption of the precursors, GDP-α-D-mannose and GDP-4-keto-6-deoxymannose, was monitored by HPLC and of L-glutamate by thinlayer chromatography (TLC). After removal of proteins and separation of the side product, α-ketoglutarate, by ion exchange chromatography and size exclusion chromatography the final product GDP-D-perosamine was obtained in 70% overall yield. The result of 1H-nuclear magnetic resonancy (NMR); 1H1H-COSY; 31P-NMR spectroscopy (Table I), of the mass spectrometry (MS) (GDP-α-D-perosamine sodium salt [(M + Na)+] 611.13 amu) and the TLC analysis of the D-perosamine component proved the structure of the product to be in accord with GDP-α-D-perosamine. Discussion The intention of this work was to identify the function of the RfbE protein by an overexpression of the rfbE gene and an in vitro production of GDP-α-D-perosamine. As yet, the specific function of a GDP-D-perosamine-synthetase has not been directly proven by in vitro assays. Rather it could been shown by mutational studies that the encoded proteins are involved in the biosynthesis of the LPS antigens (Hisatsune Table I. 1H-, 31P-NMR, chemical shifts, and coupling for GDP-D-perosamine Residue 1H/31P α-D-Perosamine H-1″ H-2″ H-3″ H-4″ H-5″ Ribose Chemical shift (p.p.m.) 5.41 3.97 3.94 3.01 4.08 Coupling constants (Hz) 3J 1″2″ 3J PH1″ 3J 2″3″ 3.5 3J 1″2″ 1.8 3J 3″4″ 10.1 3J 2″3″ 3.5 3J 3″4″ 10.1 3J 4″5″ 9.8 3J 5″6″ 6.2 3J 4″5″ 9,8 3J 5″6″ 6.2 1.8 7.1 Materials and methods H-6″ (CH3) 1.21 H-5′ a,b 4.20 H-4′ 4.31 H-3′ 4.45 3J 2′3′ 3.7 H-2′ 4.70 3J 1′2′ 6.1 3J 2′3′ 3.7 Bacterial strains, growth conditions, and media 3J 1′2′ 6.1 The bacterial strains used in this study were E. coli DH5α and E. coli BL21 (DE3) pLysS. Strains of E. coli were grown at 37°C in Luria-Bertani broth (LB) (Miller, 1972); for solid media agar-agar in a concentration of 18 g/L was added. Antibiotics were used at the following final concentrations: ampicillin 100 µg/ml, chloramphenicol 25 µg/ml. For induc- H-1′ 5.90 Base H-1′ 8.10 Phosphate P-1 –10.1 3J PP 19.6 –12.6 3J PP 19.6 3J PH-1″ P-2 658 et al., 1996; Godfroid et al., 1998; Cloeckaert et al., 2000). The RfbE protein shows homology to pyridoxal-phosphate-binding proteins (COG0399; Tatusov et al., 2001), which in part catalyze the aminogroup transfer onto deoxysugars, like NysDII (3-amino-3,6-dideoxy-D-mannose, mycosamine) in Streptomyces noursei (Brautaset et al., 2000). Therefore, as the required tools we herein evaluate the overexpressed D-perosamine biosynthetic enzymes RfbD and RfbE from V. cholerae O1, as well as the purification of GDP-D-perosaminesynthetase RfbE to homogeneity and its characterization. Surprisingly, the native and the His-tag RfbD protein were enzymatically inactive, even though they could be overexpressed in E. coli BL21 pLysS as soluble proteins. This is even though the amino acid sequence of the RfbD protein (V. cholerae) and the Gmd protein (E. coli) are 79% identical, indicating a high degree of structural congruence in the 3D structure. However, also in case of the Gmd protein of E. coli it is known that the enzyme is unstable (Sturla et al., 1997) and that the N-terminal His-tag fusion variant of the Gmd protein is also inactive (Albermann et al., 2000). Nevertheless, the recombinant Gmd protein from E. coli K12 catalyzed the formation of GDP-D-mannose to GDP-4-keto-6-deoxymannose (Sullivan et al., 1998; Albermann et al., 2000). The GDP-4-keto-6-deoxymannose, which can be synthesized this way, is used as the substrate of the GDP-4-keto-6-deoxy-D-mannose 4-aminotransferase (perosamine synthetase; RfbE). The recombinant RfbE proteins, the native and the His-tag fusion proteins, respectively, converted GDP-4-keto-6-deoxymannose in presence of L-glutamate to a new product. The NMR and MS analyses and the results of the chromatography supported that this product of the Gmd and RfbE catalyzed reactions is GDP-αD-perosamine. The in vitro preparation could be carried out efficiently in a one-step (“one-pot”) reaction system. In contrast to GDP-β-L-fucose (Sturla et al., 1997), the GDP-αD-perosamine did not show any effect of product inhibition on the activity of the GDP-D-mannose 4,6-dehydratase. Whether the GDP-α-D-perosamine exerts feedback inhibition of other biosynthetic enzymes, for instance, RfbA, RfbB, and RfbD, of V. cholerae O1 will be the subject of further investigations. In the present work, we have established a preparative enzymatic route to the synthesis of GDP-α-D-perosamine by use of the two recombinant enzymes, Gmd and RfbE, which can easily be used now for the production of GDP-α-D-perosamine, at gram scale. 7.1 Biochemicals All protein standards, antibiotics, isopropyl-β-thiogalactoside (IPTG), and nucleotides were obtained from Sigma (Deisenhofen, Germany); GDP-α-D-mannose was obtained from Calbiochem (Schwalbach, Germany). RfbE protein from V. cholerae tion of the lac operator IPTG was used at a final concentration of 24 µg/ml. Cloning of rfbD and rfbE The DNA-sequence of the rfb-gene cluster for lipopolysaccharide biosynthesis in V. cholerae O1 has been described by others (Manning et al., 1995). The genes rfbD and rfbE were amplified by PCR with Vent-polymerase (New England BioLabs, Schwalbach/Taunus, Germany). The following primers, used to amplify the genes rfbD and rfbE from the chromosomal DNA of V. cholerae O1 were designed on the basis of the published sequences and were obtained from Interactiva (Ulm, Germany): for the rfbD gene 5′-GGATATTTACATATGAATAAAAAAGTTG-3′ (forward direction); 5′-CAGGAATCATTTAAAAGATCTCACTCTAC-3′ (reverse direction); for rfbE 5′-GTGAGGTCCTTCATATGATTCCTGTAT-3′ (forward direction); 5′-GGAGGTAAGGGATCCCAAACTACTA-3′ (reverse direction). The products of the PCR amplification were further treated with NdeI and BglII restriction enzymes (Gibco BRL, Eggenstein, Germany) in the case of the rfbD fragment and with NdeI and BamHI for the rfbE PCR product, and were ligated into the vectors pET11a and pET16b (Novagen, Madison, WI), which were hydrolyzed with NdeI and BamHI, respectively (Sambrook et al., 1989). After ligation with T4DNA-ligase (BioLabs, Schwalbach/Taunus), the recombinant plasmids pET11a/rfbD (pCAW 13.1), pET11a/rfbE (pCAW 14.1), pET16b/rfbD (pCAW 13.2), and pET16b/rfbE (pCAW 14.2) were obtained and transformed into competent cells of E. coli DH5α (Hanahan, 1983). The respective cloning of the gmd gene from E. coli K12 in plasmid pCAW21.1 had been published previously (Albermann et al., 2000). DNA sequencing The insert structure of each recombinant derivative was verified by restriction analysis and DNA sequencing. The sequence of the amplified DNA was determined by the dideoxynucleotide chain-termination method (Sanger et al., 1977) using the Thermo Sequenase fluorescent labeled primer cycle sequencing kit with 7-deaza-dGTP (Amersham, Braunschweig, Germany) and an A.L.F.-Express DNA sequencer (Pharmacia, Freiburg, Germany). The sequences of both strands each were determined from the DNA of double stranded plasmids prepared by the QIAprep spin miniprep kit (Qiagen, Hilden, Germany). Overproduction of GDP-D-mannose 4,6-dehydratase (RfbD, Gmd) and GDP-4-keto-6-deoxy-D-mannose-4aminotransferase (RfbE) The recombinant plasmids (pCAW 13.1, pCAW 13.2, pCAW 14.1, pCAW 14.2, pCAW 21.1) were retransformed into competent E. coli BL21 (DE3) pLysS (Noreagen). The E. coli BL21 (DE3) pLysS cells with the recombinant plasmids were grown in LB media to an optical density of 0.6 at 540 nm. Then the cells were induced by IPTG for 90 min. Subsequently, the cells were harvested by centrifugation, washed twice in icecold 50 mM Tris–HCl buffer, pH 8, and suspended in extraction buffer (50 mM Tris–HCl buffer, pH 8, 150 mM NaCl, 20 mM MgCl2, 5 mM β-mercaptoethanol, 5 mM pyridoxal-5-phosphate). After disruption by using a 5-ml French press cell (Aminco, Silver Spring, MD) the crude extract was clarified by centrifugation at 30,000 × g for 30 min (S30). Purification of RfbE-His-tag fusion protein The RfbE His-tag fusion-protein was purified as soluble protein by affinity chromatography, using Ni-NTA-agarose (Qiagen) (Le Grice and Grueninger-Leitch, 1990; Schmitt et al., 1993). The crude extract (containing His-tag RfbE) was loaded on a Ni-NTA-agarose-column (15 mm × 60 mm) which was equilibrated with 50 mM Tris–HCl buffer, pH 8, with 300 mM NaCl, 10 mM MgCl2, and 5 mM β-mercaptoethanol. The loaded column was washed with 50 mM Tris–HCl buffer, pH 8, containing 300 mM NaCl, 10 mM MgCl2, 5 mM β-mercaptoethanol, and 20 mM imidazole. After the elution with 50 mM Tris–HCl buffer, pH 8, with 300 mM NaCl, 10 mM MgCl2, 5 mM β-mercaptoethanol, 250 mM imidazole, the proteincontaining fractions were analysed by SDS–PAGE (Laemmli, 1970) to check their purity and molecular masses. The finial purification step was carried out by gel filtration with a Sepharyl S-200 HR column (2.5 × 90 cm) (Pharmacia). The column was equilibrated with 50 mM Tris–HCl buffer, pH 8, with 10 mM MgCl2, 5 mM β-mercaptoethanol, and 300 mM NaCl. Characterization of recombinant His-tag RfbE protein The size determination of the native His-tag RfbE was performed by size exclusion chromatography on an HPLC system (Pharmacia) with a Superose 12 gel fitration column (Pharmacia). A 50 mM Tris–HCl buffer, pH 8, containing 150 mM NaCl was used as mobile phase, at a flow rate 0.5 ml/min. For the calibration curve the following proteins were applied: ovalbumin 43 kDa; bovine serum albumin 67 kDa; alcohol dehydrogenase of yeast 150 kDa; β-amylase of potato 200 kDa; apoferritin from horse spleen 443 kDa; bovine thyroglobulin 669 kDa. HPLC GMP, GDP, GTP, GDP-α-D-mannose, GDP-4-keto-6-deoxyand GDP-α-D-perosamine were separated by HPLC and detected by UV photometry (Beckman, Munich, Germany) at 260 nm. As stationary phase a reversed phase column, Eurospher ODS18, 5 µm, 250 × 4.6 mm (Knauer, Berlin, Germany) was used. As mobile phase a phosphate buffer (30 mM potassium phosphate, pH 6.0; 5 mM tetrabutylammonium hydrogensulfate, 2% acetonitrile) and acetonitrile was used (Payne and Ames, 1982). D-mannose TLC To analyze the sugar components, the nucleotide moieties were cleaved from the sugar by incubating in 1 M HCl for 10 min at 100°C. The resulting mixture and the standards of 6-deoxy-Lmannose, 6-deoxy-L-galactose, D-glucose, 2-amino-2-deoxyD-glucose, 2-amino-2-deoxy-D-galactose, 2-amino-2-deoxy-Dmannose, and D-mannose were chromatographed on TLC sheets, Silica gel F254 (Merck, Darmstadt, Germany), using a solvent system consisting of acetic acid:chloroform:water (7:6:1). All the sugar components were localized by naphthoresorcinol staining (0.2 g naphthoresorcinol in 96 ml ethanol and 4 ml concentrated sulfuric acid), aminosugars by MorganElson reagent (Chaplin and Kennedy, 1986). L-glutamate was chromatographied by the same mobile and stationary phase (see above) and detected by staining with ninhydrine (0.3 g ninhydrine in 100 ml butanol). 659 C. Albermann and W. Piepersberg Enzyme assays For the measurement of the activity of the GDP-D-mannose 4,6-dehydratase (Gmd, RfbD) each a spectroscopic (Okazaki et al., 1962; Kornfeld and Ginsburg, 1966) and chromatographic test system was used to estimate the increase of GDP4-keto-6-deoxy-D-mannose. The standard assay contained 50 mM Tris–HCl buffer, pH 8, 10 mM MgCl 2, 4 mM GDPD-mannose, 50 µM NADP+, and different amounts of GDP-Dmannose 4,6-dehydratase containing crude extract in a final volume of 100 µl. Protein concentrations were determined according to Bradford (1976). The reactions were preformed at 37°C and measured at different times between 0 and 30 min. For the photometric analysis, the reactions were stopped by adding 950 µl 100 mM NaOH to 50 µl of the reaction mixtures; the solutions were further incubated for 20 min at 37°C. After this, the absorption was measured at 320 nm (ε320nm = 2200 L mol–1 cm–1). For the HPLC analysis the samples were boiled for 1 min, and the proteins were removed by centrifugation. The enzyme activity of the GDP-4-keto-6-deoxy-D-mannose 4-aminotransferase RfbE was determined by measuring the decrease of GDP-4-keto-6-deoxy-D-mannose (see above). GDP-4-keto-6-deoxy-D-mannose was obtained by a previously published procedure (Albermann et al., 2000). The standard assay contained 50 mM Tris–HCl buffer, pH 8, 2 mM GDP-4-keto-6-deoxy-D-mannose, 20 mM MgCl2, 50 µM pyridoxal-5-phosphate, and different amounts of RfbE or His-tag RfbE and 4 mM sodium-L-glutamate. Other tested aminodonor cosubstrates (L-alanine, L-arginine, L-aspartate, L-glycine, Lglutamine, L-leucine, L-lysine, L-ornithine, L-phenylalanine, L-serine) were also added at 4 mM final concentration. The reaction was started by the addition of different concentrations of GDP-4-keto-6-deoxy-D-mannose (final volume 50 µl). Also, the increase of GDP-α-D-perosamine was observed by HPLC analysis. Enzymatic synthesis of GDP-α-D-perosamine The preparative synthesis of GDP-α-D-perosamine was carried out in a coupled enzymatic reaction by recombinant Gmd from E. coli K12 (plasmid pCAW21.1; Albermann et al., 2000) and the purified His-tag RfbE protein. The assay contained 33 µmol (20 mg) GDP-D-mannose, sodium-L-glutamate 38 µmol (7.1 mg), 50 mM Tris–HCl buffer, pH 7.5, 20 mM MgCl2, 50 µM pyridoxal phosphate, 50 µM NADP+, crude extract of E. coli BL21 pLysS pCAW21.1 and His-tag RfbE, with a GDP-D-mannose 4,6-dehydratase and GDP-4-keto-6-deoxy-Dmannose 4-aminotransferase activity each of 25 nkat in a final volume of 3 ml. This mixture was incubated for 60 min at 37°C. The proteins were removed by boiling for 1 min and subsequent centrifugation at 10,000 × g for 30 min. The time course of the reaction to GDP-α-D-perosamine was monitored by assaying small samples with HPLC analysis. Isolation of GDP-α-D-perosamine The separation of GDP-α-D-perosamine form the side products α-ketoglutarate and L-glutamate was done by an anion-exchange chromatography. This was performed with a Dowex 1 × 8 resin (mesh 200–400; formate form) (Serva, Heidelberg, Germany), elution with a linear gradient of ammonium-formate solution from 0.05 M up to 1 M in 2 h at a flow rate of 5 ml/min. GDPα-D-perosamine-containing fractions were pooled and evapo660 rated to a volume of 8 ml under reduced pressure (20 mbar) at 20–25°C. For the desalting of the preparation a gel filtration on a Sephadex G-10 column (SR 25/100; Pharmacia), with a total volume of 420 ml, was used and eluted at a flow rate of 1 ml/min. The GDP-α-D-perosamine-containing fractions were detected by UV absorption, pooled, and applied to a membrane anion exchanger Q15 (Sartorius, Göttingen, Germany) by which the GDP-α-D-perosamine preparation was equilibrated against 150 mM NaCl to yield the sodium form. After another volume reduction by evaporation to a volume of about 10 ml and a further gel filtration (Sephadex G-10 column), the sodium GDP-α-D-perosamine was lyophilized (Cryograph LCD-1, Christ, Osterrode, Germany) (yield 14 mg). The enzymatically synthesized GDP-α-D-perosamine was analyzed by NMR spectroscopy: 1H-NMR (400 MHz, D2O) δ (p.p.m.) 1.20 (d, H-6″ 3J5″6″ = 6.2 Hz); 3.01 (dd, H-4″ 3J3″4″ = 10.1 Hz, 3J4″5″ = 9.8 Hz); 3.94 (dd, H-3″ 3J3″4″ = 10.1 Hz, 3J2″3″ = 3.5 Hz); 3.97 (dd, H-2″ 3J2″3″ = 3.5 Hz, 3J1″2″ = 1.8 Hz); 4.08 (m, H-5″ 3J5″6″ = 6.2 Hz, 3J4″5″ = 9.8 Hz); 5.41 (H-1″ 3J1″2″ = 1.8 Hz, 3JPH1″ = 7.1 Hz); 4.20 (2H-5′); 4.31 (m, H-4′); 4.45 (dd, H-3′ 3J2′3′ = 3.7 Hz); 4.70 (dd, H-2′ 3J1′2′ = 6.1 Hz, 3J2′3′ = 3.7 Hz); 5.90 (dd, H-1′ 3J1′2′ = 6,1 Hz); 8.10 (s, H-1). 31P-NMR (160 MHz, D O) δ (p.p.m.) –10.1 (P-1 2J = 19.6 Hz); 2 PP –12.6 (P-2 2JPP = 19.6 Hz, 3JP-2H-1″ = 7.1 Hz). Acknowledgments We thank S. Kuberski for expert technical assistance. We are grateful to J. 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