Chemische Mikrobiologie, Bergische Universität Wuppertal, Germany
Received on January 18, 2000; revised on March 8, 2000; accepted on March 17, 2000.
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Abstract |
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Key words: GDP-ß-L-fucose/enzymatic synthesis/6-deoxyhexose/GDP-D-mannose 4,6-dehydratase/GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase-4-reductase (= GDP-ß-L-fucose synthetase)
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Introduction |
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In the case of GDP-ß-L-fucose the activated sugar metabolite is formed from mannose-6-phosphate in five steps (Figure 1). After the conversion of mannose-6-phosphate to mannose-1-phosphate, catalyzed by the mutase ManB (D-mannose-6-phosphate ketol isomerase; E.C. 5.3.1.8), and the nucleotide activation, catalyzed by the GDP--D-mannose synthase ManC (GTP:
-D-mannose-1-phosphate guanylyltransferase; E.C. 2.7.7.13), the formed intermediate GDP-
-D-mannose is processed to GDP-ß-L-fucose in three further steps which are catalyzed by two enzymes (Chang et al., 1988
; Tonetti et al., 1996
; Andrianopoulos et al., 1998
): GDP-
-D-mannose is converted into GDP-4-keto-6-deoxy-D-mannose by GDP-D-mannose 4,6-dehydratase Gmd (GDP-D-mannose 4,6-hydro-lyase; E.C. 4.2.1.47), followed by an epimerization and a final reduction step to give GDP-ß-L-fucose catalyzed by the bifunctional GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase-4-reductase WcaG (GDP-L-fucose synthetase; E.C. not yet classified).
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Results |
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Enzyme assays and HPLC-analysis
Crude ribosome-free extracts from the recombinant E.coli strains were tested for the activity of GDP-D-mannose 4,6-dehydratase and of GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase-4-reductase (GDP-L-fucose synthetase). The conversion of GDP--D-mannose to GDP-ß-L-fucose was monitored by high pressure liquid chromatography (HPLC) (Figure 4). The His-tag fusion protein variant of Gmd did not show any activity. Only the native Gmd catalyzed the dehydration of GDP-
-D-mannose. In contrast, the conversion of GDP-4-keto-6-deoxy-D-mannose to GDP-ß-L-fucose both the native WcaG protein and its His-tag fusion variant were catalytically active. In coupled reaction assays, with Gmd and WcaG or His-tag WcaG, the conversion of GDP-
-D-mannose to GDP-ß-L-fucose is low, on account of the feed-back inhibition of the GDP-D-mannose 4,6-dehydratase, after an incubation time of 90 min, only 19% of the GDP-
-D-mannose were formed to GDP-ß-L-fucose. A complete formation of GDP-ß-L-fucose, with the active Gmd and WcaG protein in the same "one-pot" assay, could be obtained, if the NADPH Co-enzyme of the GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase-4-reductase was added after the dehydration reaction to GDP-4-keto-6-deoxy-D-mannose.
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The Km and Vmax values for the substrate GDP-4-keto-6-deoxy-D-mannose were 40 µM and 23 nkat/mg protein, respectively. The Km and Vmax values for the cosubstrate NADPH were 21 µM and 10 nkat/mg protein, respectively. The reaction catalyzed by the His-tag WcaG protein has its pH optimum at 7.5 (Figure 5). Also, a slight stimulation of activity up to about 1.4- to 1.5-fold is seen after the addition of the divalent cations Ca2+ and Mg2+, an effect which is saturated at about 20 mM in both cases (cf. Figure 5).
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Preparative synthesis and isolation of GDP-ß-L-fucose
The overproduced enzyme GDP-D-mannose 4,6-dehydratase (Gmd) and the purified GDP-L-fucose synthetase (His-tag WcaG) were used for a preparative synthesis of GDP-ß-L-fucose. The synthesis was carried out by two steps starting from GDP--D-mannose (100 mg) on the way which is described in Figure 1. On account of the inhibition of GDP-D-mannose 4,6-dehydratase by GDP-ß-L-fucose it was necessary to carry out the enzymatic reaction in two steps. The formation of the products GDP-4-keto-6-deoxymannose and GDP-ß-L-fucose were followed by HPLC. 78 mg of GDP-ß-L-fucose-disodium salt were obtained after isolation by preparative HPLC and purification according to the protocol described in material and methods. The result of 13C-; 31P-; 1H-NMR-spectroscopy proved the structure of the product to be in accord with GDP-ß-fucose. Also, these data corresponded well to the published data for the chemically synthesized GDP-ß-L-fucose (Gokhale et al., 1990
; Schmidt et al., 1991
).
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Discussion |
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The in vitro preparation could not be carried out efficiently in a one-step ("one-pot") reaction system. Rather it had to be carried out by the overproduced proteins Gmd and WcaG in two successive steps. This was necessary, because of the feedback inhibition of the GDP-D-mannose 4,6-dehydratase (Gmd-protein) by the end product of this pathway GDP-ß-L-fucose (Sturla et al., 1997). In addition to the Gmd protein from E.coli we have used the overproduced Gmd protein from another enterobacterium, Yersinia enterocolitica (Zhang et al., 1996
), that shows the same effect (data not shown). Thus, enterobacterial Gmd enzymes seem to be selected for this feature, probably to prevent negative metabolic effects of an accumulation of GDP-ß-L-fucose in the cells. In the first reaction step the GDP-D-mannose is converted almost completely to GDP-4-keto-6-deoxy-D-mannose by GDP-D-mannose-4,6-dehydratase. After stopping Gmd and addition of WcaG the GDP-4-keto-6-deoxy-D-mannose can be converted to GDP-ß-L-fucose in the second reaction. This phenomenon proved the product of the WcaG-catalyzed reaction to be GDP-ß-L-fucose and not GDP-
-L-fucose, since the latter molecule is not an inhibitor of GDP-D-mannose 4,6-dehydratase (Sturla et al., 1997
).
The fact that the His-tag Gmd protein was enzymatically inactive is not surprising in view of its dinucleotide-binding pocket installed directly at its N-terminus. Obviously, the multiple His-extender peptide largely affects the protein structure in this area, perhaps by altering the accessibility of the NADP+ binding site. Such, it could prevent the normal function at least in its oxidoreductase functionality. In contrast, in the WcaG protein the His-tag does not prevent the activity of the enzyme, but it obviously prevents the formation of the quaternary structure of a homodimeric enzyme which is observed in case of the native WcaG protein (Rizzi et al., 1998). Thus, the multiple His-extender peptide seems to lead to a monomer form of the GDP-ß-L-fucose synthetase, which, however, seems not to be affected in its activity as proven by the correspondence in kinetic data obtained with both enzyme forms.
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Materials and methods |
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Bacterial strains, growth conditions, and media
The bacterial strains used in this study were Escherichia coli DH5 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 agaragar in a concentration of 18 g/l was added. Antibiotics were used at the following finial concentrations: ampicillin 100 µg/ml, chloramphenicol 25 µg/ml. For induction of the lac operator IPTG was used at a finial concentration of 24 µg/ml.
Cloning of gmd and wcaG
The DNA-sequence of the wca-gene cluster for the colanic acid biosynthesis in E.coli K12 was described (Stevenson et al., 1996). The genes gmd and wcaG were amplified by PCR with Vent polymerase (BioLabs, Schwalbach/Taunus). Chromosomal DNA was prepared from strain E.coli DH5
as described previously (Wilson, 1988
). The following primers, used to amplify the genes gmd and wcaG, were designed on the basis of the published sequence (Stevenson et al., 1996
) and were obtained from MWG (Ebersberg, Germany): for the gmd gene 5'ACAGAGGAATAACATATGTCAAAAGTCGC3' (forward direction); 5'CCAGCAATAAAAGATCTTTGTTTACTCATGC3' (reverse direction); for wcaG 5'ATCGCGCTGGAGTCATACATATGAGTAAAC3' forward; 5'ACGTAAAAAGATCTTTACCCCCGAAA3' reverse; for gmd/wcaG 5'ACAGAGGAATAACATATGTCAAAAGTCGC3' forward; 5'ACGTAAAAAGATCTTTACCCCCGAAA3' reverse. The products of the PCR amplification were digested with NdeI and BglII restriction enzymes (Gibco BRL, Eggenstein, Germany) and ligated (Sambrook et al., 1989
) into the vectors pET11a and pET16b (Novagen, Madison, WI) which were hydrolyzed with NdeI and BamHI. After ligation with T4-DNA-ligase (BioLabs, Schwalbach/Taunus), the recombinant plasmids pET11a/gmd (pCAW21.1), pET11a/wcaG (pCAW22.1), pET11a/gmd-wcaG (pCAW23), pET16b/gmd (pCAW21.2), pET16b/wcaG (pCAW22.2) were obtained and transformed into competent cells of E.coli DH5
(Hanahan, 1983
). The insert structure of each recombinant derivative was verified by restriction analysis and DNA-sequencing. For over-expression of the proteins the recombinant plasmids were retransformed into competent E.coli BL21 (DE3) pLysS (Cohen et al., 1972
).
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 sequence of both strands 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 (Gmd) and GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase-4-reductase (WcaG)
For the overproduction of Gmd and WcaG, 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. The cells were then induced by IPTG for 90 min. Subsequently, the cells were harvested by centrifugation, washed twice in ice-cold 50 mM Tris/HCl-buffer pH 7.5, and suspended in extraction buffer (50 mM Tris/HCl-buffer pH 7.5, 150 mM NaCl, 10 mM MgCl2, 5 mM ß-mercaptoethanol, 5 mM EDTA). After the disruption by sonication the crude extract was clarified by centrifugation at 30,000 x g for 30 min (S30).
Purification of His-tag fusion proteins
The His-tag Gmd and the His-tag WcaG fused proteins were purified as soluble protein by affinity chromatography, using Ni-NTA-agarose (Qiagen, Hilden, Germany) (Le Grice and Grueninger-Leitch, 1990; Schmitt et al., 1993
). The crude extract (containing His-tag WcaG or His-tag Gmd) was loaded on a Ni-NTA-agarose-column (15 mm x 60 mm) which was equilibrated with 50 mM Tris/HCl-buffer pH 7.5 with 150 mM NaCl, 10 mM MgCl2, and 5 mM ß-mercaptoethanol. The loaded column was washed with 50 mM Tris/HCl-buffer pH 7.5 containing 150 mM NaCl, 10 mM MgCl2, 5 mM ß-mercaptoethanol, and 20 mM imidazol. After the elution with 50 mM Tris/HCl-buffer pH 7.5 with 10 mM MgCl2, 5 mM ß-mercaptoethanol, 200 mM imidazol, and 10% glycerol the protein-containing fractions were analyzed by SDSPAGE (Laemmli, 1970
) to check the purity and the molecular mass. Subsequently the imidazol was removed by gel filtration with a PD-10 column (Pharmacia, Freiburg, Germany), the column was equilibrated with 50 mM Tris/HCl-buffer pH 7.5 with 10 mM MgCl2, 5 mM ß-mercaptoethanol, 150 mM NaCl, and 10% glycerol.
Characterization of recombinant WcaG
The size of the native His-tag WcaG was performed by size exclusion chromatography on a FPLC system (Pharmacia, Freiburg) with a Superose 12 gel filtration column (Pharmacia, Freiburg). As mobile phase was used a 50 mM Tris/HCl-buffer pH 7.5 containing 150 mM NaCl, at a flow rate 0.5 ml/min. For the calibration curve the following proteins were applied: ribonuclease 13.7 kDa, chymotrypsinogen A 25 kDa, ovalbumin 43 kDa, bovine serum albumin 67 kDa, alcohol dehydrogenase 150 kDa.
High pressure liquid chromatography (HPLC)
NADP+, NADPH, guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), GDP-D-mannose, GDP-4-keto-6-deoxy-D-mannose, and GDP-ß-L-fucose were separated by HPLC and detected by UV-photometry (Beckman, München, Germany) at 260 nm. As mobile phase a phosphate-buffer (30 mM potassium phosphate, pH 6.0; 5 mM tetrabutylammonium hydrogen sulfate, 2% acetonitrile) and acetonitrile was used. As stationary phase a reversed phase column Eurospher ODS18, 5 µm, 250 x 4.6 mm (Knauer, Berlin, Germany) was used (Payne and Ames, 1982).
Enzyme assays
For the measurement of the activity of the GDP-D-mannose 4,6-dehydratase, a spectroscopic (Okazaki et al., 1962; Kornfeld and Ginsburg, 1966
) and chromatographic test system was used to estimated the increase of GDP-4-keto-6-deoxy-D-mannose. The standard assay contained 50 mM Tris/HCl-buffer pH 7.5, 10 mM MgCl2, 4 mM GDP-D-mannose, 50 µM NADP+ and different amounts of Gmd or His-tag Gmd-containing crude extract in a finial volume of 100 µl. Protein concentration was determined according to Bradford (1976)
. The reactions were performed at 37°C and measured at different times between 0 and 60 min. For the photometric analysis, the reaction were stopped by adding 950 µl 100 mM NaOH to 50 µl of the reaction mixture, the solution was further incubated for 20 min at 37°C. After this, the absorption was measured at 320 nm. For the HPLC analysis the probes were boiled and the proteins were removed by centrifugation. The enzyme activity of the GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase-4-reductase was determined by measuring the consumption of NADPH at 340 nm and 37°C. The standard assay contained 50 mM Tris/HCl-buffer pH 7.5, 2 mM GDP-4-keto-6-deoxy-D-mannose, 10 mM MgCl2, different amounts of WcaG or His-tag WcaG and 4 mM NADPH. The reaction was started by the addition of different concentrations of GDP-4-keto-6-deoxy-D-mannose (finial volume 500 µl). Also, the increase of GDP-ß-L-fucose was observed by HPLC analysis.
Synthesis of GDP-ß-L-fucose
The first step of the preparative synthesis of GDP-ß-L-fucose is the conversion of GDP-D-mannose to GDP-4-keto-6-deoxy-D-mannose. The preparative enzyme assay contains 165 µmol (100 mg) GDP-D-mannose, 50 mM Tris/HCl-buffer pH 7.5, 10 mM MgCl2, crude extract of E.coli BL21 pLysS pCAW21.1 with a GDP-D-mannose 4,6-dehydratase activity of 120 nkat in a finial volume of 6 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 x g for 30 min. The second step of conversion was started by the addition of 200 µmol NADPH and 120 nkat GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase-4-reductase (His-tag WcaG) to the supernatant. Subsequently, the mixture was incubated for a second time at 37°C for 60 min. The GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase-4-reductase was also separated by boiling and subsequent centrifugation. The course of each reaction to GDP-ß-L-fucose was monitored by HPLC analysis.
Isolation of GDP-ß-L-fucose
The purification of GDP-ß-L-fucose from the side products NADP+ and NADPH was done by a preparative HPLC. The chromatography was performed with a Eurospher ODS18 (20 x 250 mm) column (Knauer, Berlin, Germany) under the same conditions as for the analytical chromatography (see High pressure liquid chromatography [HPLC]). GDP-L-fucose-containing fractions were pooled and evaporated to a volume of 12 ml under reduced pressure (20 mbar) at 2025°C. For the desalting of the preparation a gel filtration on a Sephadex G-10 column (SR 25/100; Pharmacia, Freiburg, Germany), with a total volume of 398 ml, was used and eluted at a flow rate of 1 ml/min. The GDP-ß-L-fucose-containing fractions were detected by UV-absorption, pooled and applied to a membrane anion exchanger Q15 (Sartorius, Göttingen, Germany) by which the GDP-ß-L-fucose preparation was equilibrated against 150 mM NaCl to yield the Na+ form. After another volume reduction by evaporation to a volume of about 12 ml and a further gel filtration (Sephadex G-10 column), the sodium GDP-ß-L-fucose was lyophilized (Cryograph LCD-1, Christ, Osterrode, Germany) (yield 78 mg).
The enzymatically synthesized GDP-ß-L-fucose was analyzed by NMR-spectroscopy: 1H-NMR (400 MHz, D2O) (p.p.m.) = 1,1 (d, H-6'', 3J5''6''= 6,4 Hz); 3,55 (dd H-2'', 3J2''3'' = 11 Hz, 3J1''2''= 8,1 Hz); 3,65 (dd, H-3''; 3J3''4'' = 3,5 Hz, 3J2''3'' = 11 Hz); 3,71 (m, H-4'', 3J3''4'' = 3,5); 3,75 (m, H-5''); 4,2 (m, 2H, H-5a'H-5b'); 4,52 (m, H-4'); 4,59 (dd, H-3'); 4,8 (dd, H-2', 3J1'2'= 6,1 Hz, 3J2'3'= 3,7 Hz); 4,91 (dd, H-1'', 3J1''2'' = 8,1 Hz,3JP-2H1'' = 8,3); 5,9 (d, H-1', 3J1'2'= 6,1 Hz ); 8,1 (s, H-1).
13C-NMR (400 MHz, D2O) (p.p.m.) = 17,5 C'-6''; 33,2 C-2'; 64,6 C-5'; 73,1 73,2 72,5 C-3'' C-4'' C-5'; 74,5 C-3''; 75,8 C-2''; 100,4 C-1''; 118,3 C-5; 139,6 C-1; 153,9 C-4; 156,2 C-2; 161,2 C-6.
31P-NMR (400 MHz, D2O) (p.p.m.) = 9,7 (P-1, 2JPP = 19,9 Hz); 11,5 (P-2, 2Jpp = 19,9 Hz, 3JP-2H-1''= 8,3 Hz).
For reference, the published 1H-NMR data of bis(triethylammonium)-ß-L-fucopyranosyl guanosine-5'-pyrophosphate could be used (Gokhale et al., 1990; Schmidt et al., 1991
).
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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