Differential role of NADP+ and NADPH in the activity and structure of GDP-D-mannose 4,6-dehydratase from two chlorella viruses

GDP-D-mannose 4,6-dehydratase (GMD) is a key enzyme involved in the synthesis of 6-deoxyhexoses in prokaryotes and eukaryotes. Paramecium bursaria chlorella virus-1 (PBCV-1) encodes a functional GMD, which is unique among characterized GMDs because it also has a strong stereospecific NADPH-dependent reductase activity leading to GDP-D-rhamnose formation (Tonetti, M., Zanardi, D., Gurnon, J., Fruscione, F., Armirotti, A., Damonte, G., Sturla, L., De Flora, A., and Van Etten, J.L. (2003) J. Biol. Chem. 278, 21559-21565). In the present study we characterized a recombinant GMD encoded by another chlorella virus, Acanthocystis turfacea chlorella virus 1 (ATCV-1), demonstrating that it has the expected dehydratase activity. However, it also displayed significant differences when compared with PBCV-1 GMD. In particular, ATCV-1 GMD lacks the reductase activity present in the PBCV-1 enzyme. Using recombinant PBCV-1 and ATCV-1 GMDs, we determined that the enzymatically active proteins contain tightly bound NADPH and that NADPH is essential for maintaining the oligomerization status as well as for the stabilization and function of both enzymes. Phylogenetic analysis indicates that PBCV-1 GMD is the most evolutionary diverged of the GMDs. We conclude that this high degree of divergence was the result of the selection pressures that led to the acquisition of new reductase activity to synthesize GDP-D-rhamnose while maintaining the dehydratase activity in order to continue to synthesize GDP-L-fucose.     

An epimerase-reductase in L-fucose synthesis

The first committed enzyme in GDP-L-fucose formation from GDP-D-mannose is GDP-D-mannose 4,6-dehydratase, which forms GDP-4-keto-6-deoxy-D-mannose. The uncertain enzymatic steps beyond this point were examined in this study. Assays were developed for the epimerase and reductase activities which the putative pathway would predict. A protein was isolated exhibiting homogeneity by several criteria. This single protein, which forms GDP-L-fucose from GDP-4-keto-6-deoxy-D-mannose and NADH, appears to possess both epimerase and reductase capabilities and may be termed GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase. Analysis on a molecular sieve column using fast protein liquid chromatography established a molecular weight of 63,100 for the native enzyme, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis established a subunit molecular weight of 31,500.     

Functional expression of Pseudomonas aeruginosa GDP-4-keto-6-deoxy-D-mannose reductase which synthesizes GDP-rhamnose.

Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium that causes severe infections in a number of hosts from plants to mammals. A-band lipopolysaccharide of P. aeruginosa contains d-rhamnosylated O-antigen. The synthesis of GDP-D-rhamnose, the d-rhamnose donor in d-rhamnosylation, starts from GDP-D-mannose. It is first converted by the GDP-mannose-4,6-dehydratase (GMD) into GDP-4-keto-6-deoxy-D-mannose, and then reduced to GDP-D-rhamnose by GDP-4-keto-6-deoxy-D-mannose reductase (RMD). Here, we describe the enzymatic characterization of P. aeruginosa RMD expressed in Saccharomyces cerevisiae. Previous success in functional expression of bacterial gmd genes in S. cerevisiae allowed us to convert GDP-D-mannose into GDP-4-keto-6-deoxy-D-mannose. Thus, coexpression of the Helicobacter pylori gmd and P. aeruginosa rmd genes resulted in conversion of the 4-keto-6-deoxy intermediate into GDP-deoxyhexose. This synthesized GDP-deoxyhexose was confirmed to be GDP-rhamnose by HPLC, matrix-assisted laser desorption/ionization time-of-flight MS, and finally NMR spectroscopy. The functional expression of P. aeruginosa RMD in S. cerevisiae will provide a tool for generating GDP-rhamnose for in vitro rhamnosylation of glycoprotein and glycopeptides.     

Quaternary assembly and crystal structure of GDP-D-mannose 4,6 dehydratase from Paramecium bursaria Chlorella virus

GDP-D-mannose 4,6 dehydratase is the first enzyme in the de novo biosynthetic pathway of GDP-L-fucose, the activated form of L-fucose, a monosaccharide found in organisms ranging from bacteria to mammals. We determined the three-dimensional structure of GDP-D-mannose 4,6 dehydratase from the Paramecium bursaria Chlorella virus at 3.8A resolution. Unlike other viruses that use the host protein machinery to glycosylate their proteins, P. bursaria Chlorella virus modifies its structural proteins using many glycosyltransferases, being the first virus known to encode enzymes involved in sugar metabolism. P. bursaria Chlorella virus GDP-D-mannose 4,6 dehydratase belongs to the short-chain dehydrogenase/reductase protein superfamily. Accordingly, the family fold and the specific Thr, Tyr, and Lys catalytic triad are well conserved in the viral enzyme.     

Preparative synthesis of GDP-beta-L-fucose by recombinant enzymes from enterobacterial sources

The 6-deoxyhexose L-fucose is an important and characteristic element in glycoconjugates of bacteria (e.g., lipopolysaccharides), plants (e.g., xyloglucans) and animals (e.g., glycolipids, glycoproteins, and oligosaccharides). The biosynthetic pathway of GDP-L-fucose starts with a dehydration of GDP-D-mannose catalyzed by GDP-D-mannose 4,6-dehydratase (Gmd) creating GDP-4-keto-6-deoxymannose which is subsequently converted by the GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase-4-reductase (WcaG; GDP-beta-L-fucose synthetase) to GDP-beta-L-fucose. Both biosynthetic genes gmd and wcaG were cloned from Escherichia coli K12 and the enzymes overexpressed under control of the T7 promoter in the expression vectors pET11a and pET16b, yielding both native and N-terminal His-tag fusion proteins, respectively. The activities of the Gmd and WcaG were analyzed. The enzymatic conversion from GDP-D-mannose to GDP-beta-L-fucose was optimized and the final product was purified. The formation of GDP-beta-L-fucose by the recombinant enzymes was verified by HPLC and NMR analyses. The His-tag fusion variants of the Gmd and WcaG proteins were purified to near homogeneity. The His-tag Gmd recombinant enzyme was inactive, whereas His-tag WcaG showed very similar enzymatic properties relative to the native GDP-beta-L-fucose synthetase. With the purified His-tag WcaG Km and Vmax values, respectively, of 40 microM and 23 nkat/mg protein for the substrate GDP-4-keto-6-deoxy-D-mannose and of 21 microM and 10 nkat/mg protein for the cosubstrate NADPH were obtained; a pH optimum of 7.5 was determined and the enzyme was stimulated to equal extend by the divalent cations Mg2+ and Ca2+. The Gmd enzyme showed a strong feedback inhibition by GDP-beta-L-fucose.     

Synthesis of the milk oligosaccharide 2'-fucosyllactose using recombinant bacterial enzymes

The enzymatic synthesis of GDP-beta-L-fucose and its enzymatic transfer reaction using recombinant enzymes from bacterial sources was examined. The GDP-D-mannose 4,6-dehydratase and the GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase-4-reductase from Escherichia coli K-12, respectively, were used to catalyse the conversion of GDP-alpha-D-mannose to GDP-beta-L-fucose with 78% yield. For the transfer of the L-fucose to an acceptor, we cloned and overproduced the alpha-(1-->2)-fucosyltransferase (FucT2) protein from Helicobacter pylori. We were able to synthesise 2'-fucosyllactose using the overproduced FucT2 enzyme, enzymatically synthesised GDP-L-fucose and lactose. The isolation of 2'-fucosyllactose was accomplished by anion-exchange chromatography and gel filtration to give 65% yield.     

Cloning and functional expression of a novel GDP-6-deoxy-D-talose synthetase from Actinobacillus actinomycetemcomitans

Actinobacillus actinomycetemcomitans is a Gram-negative coccobacillus that can cause various forms of severe periodontitis and other nonoral infections in human patients. The serotype a-specific polysaccharide antigen of A. actinomycetemcomitans contains solely 6-deoxy-D-talose and its O-2 acetylated modification. This polysaccharide is synthesized from the donor GDP-6-deoxy-D-talose with the relevant talosylation enzyme(s). In the synthesis of GDP-6- deoxy-D-talose, GDP-D-mannose is first converted by GDP-mannose-4,6-dehydratase (GMD) to GDP-4-keto-6-deoxy-D-mannose and then reduced to GDP-6-deoxy-D-talose by GDP-6-deoxy-D-talose synthetase (GTS). In this study, we cloned and overexpressed in Escherichia coli the A. actinomycetemcomitans GTS enzyme responsible for the synthesis of GDP-6-deoxy-D-talose. The recombinant A. actinomycetemcomitans GTS enzyme expressed in E. coli converted the GDP-4-keto-6-deoxy-intermediate to a novel GDP-deoxyhexose. The synthesized GDP-deoxyhexose was shown to be GDP-6-deoxy-D-talose by HPLC, MALDI-TOF MS, and NMR spectroscopy. The functional expression of gts provides another enzymatically defined pathway for the synthesis of GDP-deoxyhexoses, which can be used as donors for the corresponding glycosyltransferases.     

Functional expression of Escherichia coli enzymes synthesizing GDP-L-fucose from inherent GDP-D-mannose in Saccharomyces cerevisiae

Fucosylation of glycans on glycoproteins and -lipids requires the enzymatic activity of relevant fucosyltransferases and GDP-L-fucose as the donor. Due to the biological importance of fucosylated glycans, a readily accessible source of GDP-L-fucose would be required. Here we describe the construction of a stable recombinant S.cerevisiae strain expressing the E.coli genes gmd and wcaG encoding the two enzymes, GDP-mannose-4,6-dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase/4-reductase (GMER(FX)) respectively, needed to convert GDP-mannose to GDP-fucose via the de novo pathway. Taking advantage of the rich inherent cytosolic GDP-mannose pool in S.cerevisiae cells we could easily produce 0.2 mg/l of GDP-L-fucose with this recombinant yeast strain without addition of any external GDP-mannose. The GDP-L-fucose product could be used as the fucose donor for alpha1,3fucosyltransferase to synthesize sialyl Lewis x (sLex), a glycan crucial for the selectin-dependent leukocyte traffic.     

Composition of Drosophila melanogaster proteome involved in fucosylated glycan metabolism.

The whole genome approach enables the characterization of all components of any given biological pathway. Moreover, it can help to uncover all the metabolic routes for any molecule. Here we have used the genome of Drosophila melanogaster to search for enzymes involved in the metabolism of fucosylated glycans. Our results suggest that in the fruit fly GDP-fucose, the donor for fucosyltransferase reactions, is formed exclusively via the de novo pathway from GDP-mannose through enzymatic reactions catalyzed by GDP-D-mannose 4,6-dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase/4-reductase (GMER, also known as FX in man). The Drosophila genome does not have orthologs for the salvage pathway enzymes, i.e. fucokinase and GDP-fucose pyrophosphorylase synthesizing GDP-fucose from fucose. In addition we identified two novel fucosyltransferases predicted to catalyze alpha1,3- and alpha1,6-specific linkages to the GlcNAc residues on glycans. No genes with the capacity to encode alpha1,2-specific fucosyltransferases were found. We also identified two novel genes coding for O-fucosyltransferases and a gene responsible for a fucosidase enzyme in the Drosophila genome. Finally, using the Drosophila CG4435 gene, we identified two novel human genes putatively coding for fucosyltransferases. This work can serve as a basis for further whole-genome approaches in mapping all possible glycosylation pathways and as a basic analysis leading to subsequent experimental studies to verify the predictions made in this work.     

Biochemical and Functional Studies on the Burkholderia cepacia Complex bceN Gene,Encoding a GDP-D-Mannose 4,6-Dehydratase

This work reports the biochemical and functional analysis of the Burkholderia cenocepacia J2315 bceN gene, encoding a protein with GDP-D-mannose 4,6-dehydratase enzyme activity (E.C.4.2.1.47). Data presented indicate that the protein is active when in the tetrameric form, catalyzing the conversion of GDP-D-mannose into GDP-4-keto-6-deoxy-D-mannose. This sugar nucleotide is the intermediary necessary for the biosynthesis of GDP-D-rhamnose, one of the sugar residues of cepacian, the major exopolysaccharide produced by environmental and human, animal and plant pathogenic isolates of the Burkholderia cepacia complex species. V_max and K_m values of 1.5±0.2 µmol.min⁻¹.mg⁻¹ and 1024±123 µM, respectively, were obtained from the kinetic characterization of the B. cenocepacia J2315 BceN protein by NMR spectroscopy, at 25°C and in the presence of 1 mol MgCl₂ per mol of protein. The enzyme activity was strongly inhibited by the substrate, with an estimated K_i of 2913±350 µM. The lack of a functional bceN gene in a mutant derived from B. cepacia IST408 slightly reduced cepacian production. However, in the B. multivorans ATCC17616 with bceN as the single gene in its genome with predicted GMD activity, a bceN mutant did not produce cepacian, indicating that this gene product is required for cepacian biosynthesis.     

Characterization of the GDP-D-mannose biosynthesis pathway in Coxiella burnetii: the initial steps for GDP-β-D-virenose biosynthesis

Coxiella burnetii, the etiologic agent of human Q fever, is a gram-negative and naturally obligate intracellular bacterium. The O-specific polysaccharide chain (O-PS) of the lipopolysaccharide (LPS) of C. burnetii is considered a heteropolymer of the two unusual sugars β-D-virenose and dihydrohydroxystreptose and mannose. We hypothesize that GDP-D-mannose is a metabolic intermediate to GDP-β-D-virenose. GDP-D-mannose is synthesized from fructose-6-phosphate in 3 successive reactions; Isomerization to mannose-6-phosphate catalyzed by a phosphomannose isomerase (PMI), followed by conversion to mannose-1-phosphate mediated by a phosphomannomutase (PMM) and addition of GDP by a GDP-mannose pyrophosphorylase (GMP). GDP-D-mannose is then likely converted to GDP-6-deoxy-D-lyxo-hex-4-ulopyranose (GDP-Sug), a virenose intermediate, by a GDP-mannose-4,6-dehydratase (GMD). To test the validity of this pathway in C. burnetii, three open reading frames (CBU0671, CBU0294 and CBU0689) annotated as bifunctional type II PMI, as PMM or GMD were functionally characterized by complementation of corresponding E. coli mutant strains and in enzymatic assays. CBU0671, failed to complement an Escherichia coli manA (PMM) mutant strain. However, complementation of an E. coli manC (GMP) mutant strain restored capsular polysaccharide biosynthesis. CBU0294 complemented a Pseudomonas aeruginosa algC (GMP) mutant strain and showed phosphoglucomutase activity (PGM) in a pgm E. coli mutant strain. Despite the inability to complement a manA mutant, recombinant C. burnetii PMI protein showed PMM enzymatic activity in biochemical assays. CBU0689 showed dehydratase activity and determined kinetic parameters were consistent with previously reported data from other organisms. These results show the biological function of three C. burnetii LPS biosynthesis enzymes required for the formation of GDP-D-mannose and GDP-Sug. A fundamental understanding of C. burnetii genes that encode PMI, PMM and GMP is critical to fully understand the biosynthesic pathway of GDP-β-D-virenose and LPS structure in C. burnetii.     

Probing the catalytic mechanism of GDP-4-keto-6-deoxy-d-mannose Epimerase/Reductase by kinetic and crystallographic characterization of site-specific mutants

GDP-4-keto-6-deoxy-d-mannose epimerase/reductase is a bifunctional enzyme responsible for the last step in the biosynthesis of GDP-l-fucose, the substrate of fucosyl transferases. Several cell-surface antigens, including the leukocyte Lewis system and cell-surface antigens in pathogenic bacteria, depend on the availability of GDP-l-fucose for their expression. Therefore, the enzyme is a potential target for therapy in pathological states depending on selectin-mediated cell-to-cell interactions. Previous crystallographic investigations have shown that GDP-4-keto-6-deoxy-d-mannose epimerase/reductase belongs to the short-chain dehydrogenase/reductase protein homology family. The enzyme active-site region is at the interface of an N-terminal NADPH-binding domain and a C-terminal domain, held to bind the substrate. The design, expression and functional characterization of seven site-specific mutant forms of GDP-4-keto-6-deoxy-d-mannose epimerase/reductase are reported here. In parallel, the crystal structures of the native holoenzyme and of three mutants (Ser107Ala, Tyr136Glu and Lys140Arg) have been investigated and refined at 1. 45-1.60 A resolution, based on synchrotron data (R-factors range between 12.6 % and 13.9 %). The refined protein models show that besides the active-site residues Ser107, Tyr136 and Lys140, whose mutations impair the overall enzymatic activity and may affect the coenzyme binding mode, side-chains capable of proton exchange, located around the expected substrate (GDP-4-keto-6-deoxy-d-mannose) binding pocket, are selectively required during the epimerization and reduction steps. Among these, Cys109 and His179 may play a primary role in proton exchange between the enzyme and the epimerization catalytic intermediates. Finally, the additional role of mutated active-site residues involved in substrate recognition and in enzyme stability has been analyzed.     

Expression and identification of the RfbE protein from Vibrio cholerae O1 and its use for the enzymatic synthesis of GDP-D-perosamine

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 fructose-6-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-6-deoxy-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.     

Biochemical characterization of GDP-l-fucose de novo synthesis pathway in fungus Mortierella alpina

Mortierella alpina is a filamentous fungus commonly found in soil, which is able to produce large amount of polyunsaturated fatty acids. l-Fucose is an important sugar found in a diverse range of organisms, playing a variety of biological roles. In this study, we characterized the de novo biosynthetic pathway of GDP-l-fucose (the nucleotide-activated form of l-fucose) in M. alpina. Genes encoding GDP-d-mannose 4,6-dehydratase (GMD) and GDP-keto-6-deoxymannose 3,5-epimerase/4-reductase (GMER) were expressed heterologously in Escherichia coli. The recombinant enzymes were produced as His-tagged fusion proteins. Conversion of GDP-mannose to GDP-4-keto-6-deoxy mannose by GMD and GDP-4-keto-6-deoxy mannose to GDP-l-fucose by GMER were analyzed by capillary electrophoresis, electro-spray ionization-mass spectrometry, and nuclear magnetic resonance spectroscopy. The k_m values of GMD for GDP-mannose and GMER for GDP-4-keto-6-deoxy mannose were determined to be 0.77 mM and 1.047 mM, respectively. Both NADH and NADPH may be used by GMER as the coenzyme. The optimum temperature and pH were determined to be 37 °C and pH 9.0 (GMD) or pH 7.0 (GMER). Divalent cations are not required for GMD and GMER activity, and the activities of both enzymes may be enhanced by DTT. To our knowledge this is the first report on the characterization of GDP-l-fucose biosynthetic pathway in fungi.     

Structure of the MUR1 GDP-mannose 4,6-dehydratase from Arabidopsis thaliana: implications for ligand binding and specificity

GDP-D-mannose 4,6-dehydratase catalyzes the first step in the de novo synthesis of GDP-L-fucose, the activated form of L-fucose, which is a component of glycoconjugates in plants known to be important to the development and strength of stem tissues. We have determined the three-dimensional structure of the MUR1 dehydratase isoform from Arabidopsis thaliana complexed with its NADPH cofactor as well as with the ligands GDP and GDP-D-rhamnose. MUR1 is a member of the nucleoside-diphosphosugar modifying subclass of the short-chain dehydrogenase/reductase enzyme family, having homologous structures and a conserved catalytic triad of Lys, Tyr, and Ser/Thr residues. MUR1 is the first member of this subfamily to be observed as a tetramer, the interface of which reveals a close and intimate overlap of neighboring NADP(+)-binding sites. The GDP moiety of the substrate also binds in an unusual syn conformation. The protein-ligand interactions around the hexose moiety of the substrate support the importance of the conserved triad residues and an additional Glu side chain serving as a general base for catalysis. Phe and Arg side chains close to the hexose ring may serve to confer substrate specificity at the O2 position. In the MUR1/GDP-D-rhamnose complex, a single unique monomer within the protein tetramer that has an unoccupied substrate site highlights the conformational changes that accompany substrate binding and may suggest the existence of negative cooperativity in MUR1 function.     

GDP-4-keto-6-deoxy-D-mannose 3-dehydratase, accommodating a sugar substrate in the active site

Colitose is a dideoxysugar found in the O-antigen of the lipopolysaccharide that coats the outer membrane of some Gram-negative bacteria. Four enzymes are required for its production starting from D-mannose-1-phosphate and GTP. The focus of this investigation is GDP-4-keto-6-deoxy-D-mannose 3-dehydratase or ColD, which catalyzes the removal of the C3'-hydroxyl group from GDP-4-keto-6-deoxymannose. The enzyme is pyridoxal 5'-phosphate-dependent, but unlike most of these proteins, the conserved lysine residue that covalently holds the cofactor in the active site is replaced with a histidine residue. Here we describe the three-dimensional structure of ColD, determined to 1.7A resolution, whereby the active site histidine has been replaced with an asparagine residue. For this investigation, crystals of the site-directed mutant protein were grown in the presence of GDP-4-amino-4,6-dideoxy-D-mannose (GDP-perosamine). The electron density map clearly reveals the presence of the sugar analog trapped in the active site as an external aldimine. The active site is positioned between the two subunits of the dimer. Whereas the pyrophosphoryl groups of the ligand are anchored to the protein via Arg-219 and Arg-331, the hydroxyl groups of the hexose only lie within hydrogen bonding distance to ordered water molecules. Interestingly, the hexose moiety of the ligand adopts a boat rather than the typically observed chair conformation. Activity assays demonstrate that this mutant protein cannot catalyze the dehydration step. Additionally, we report data revealing that wild-type ColD is able to catalyze the production of GDP-4-keto-3,6-dideoxymannose using GDP-perosamine instead of GDP-4-keto-6-deoxymannose as a substrate.     

Structural and kinetic analysis of Escherichia coli GDP-mannose 4,6 dehydratase provides insights into the enzyme's catalytic mechanism and regulation by GDP-fucose

Background: GDP-mannose 4,6 dehydratase (GMD) catalyzes the conversion of GDP-(D)-mannose to GDP-4-keto, 6-deoxy-(D)-mannose. This is the first and regulatory step in the de novo biosynthesis of GDP-(L)-fucose. Fucose forms part of a number of glycoconjugates, including the ABO blood groups and the selectin ligand sialyl Lewis X. Defects in GDP-fucose metabolism have been linked to leukocyte adhesion deficiency type II (LADII). Results: The structure of the GDP-mannose 4,6 dehydratase apo enzyme has been determined and refined using data to 2.3 A resolution. GMD is a homodimeric protein with each monomer composed of two domains. The larger N-terminal domain binds the NADP(H) cofactor in a classical Rossmann fold and the C-terminal domain harbors the sugar-nucleotide binding site. We have determined the GMD dissociation constants for NADP, NADPH and GDP-mannose. Each GMD monomer binds one cofactor and one substrate molecule, suggesting that both subunits are catalytically competent. GDP-fucose acts as a competitive inhibitor, suggesting that it binds to the same site as GDP-mannose, providing a mechanism for the feedback inhibition of fucose biosynthesis. Conclusions: The X-ray structure of GMD reveals that it is a member of the short-chain dehydrogenase/reductase (SDR) family of proteins. We have modeled the binding of NADP and GDP-mannose to the enzyme and mutated four of the active-site residues to determine their function. The combined modeling and mutagenesis data suggests that at position 133 threonine substitutes serine as part of the serine-tyrosine-lysine catalytic triad common to the SDR family and Glu 135 functions as an active-site base.     

A bifunctional epimerase-reductase acts downstream of the MUR1 gene product and completes the de novo synthesis of GDP-L-fucose in Arabidopsis

L-Fucose is a monosaccharide found as a component of glycoproteins and cell wall polysaccharides in higher plants. The MUR1 gene of Arabidopsis thaliana encodes a GDP-D-mannose 4,6-dehydratase catalyzing the first step in the de novo synthesis of GDP-L-fucose from GDP-D-mannose (Bonin et al. 1997, Proc. Natl Acad. Sci. USA, 94, 2085-2090). Plant genes encoding the subsequent steps in L-fucose synthesis (3,5-epimerization and 4-reduction) have not been described previously. Based on sequence similarities to a bacterial gene involved in capsule synthesis we have cloned a gene from Arabidopsis, now designated GER1, which encodes a bifunctional 3, 5-epimerase-4-reductase in L-fucose synthesis. The combined action of the MUR1 and GER1 gene products converts GDP-D-mannose to GDP-L-fucose in vitro demonstrating that this entire nucleotide-sugar interconversion pathway could be reconstituted using plant genes expressed in Escherichia coli. In vitro assays indicated that the GER1 protein does not act as a GDP-D-mannose 3, 5-epimerase, an enzymatic activity involved in the de novo synthesis of GDP-L-galactose and L-ascorbic acid. Similarly, L-ascorbate levels in GER1 antisense plants were unchanged indicating that GDP-D-mannose 3,5-epimerase is encoded by a separate gene.     

Mutation in GDP-fucose synthesis genes of Sinorhizobium fredii alters Nod factors and significantly decreases competitiveness to nodulate soybeans

We mutagenized Sinorhizobium fredii HH103-1 with Tn5-B20 and screened about 2,000 colonies for increased beta-galactosidase activity in the presence of the flavonoid naringenin. One mutant, designated SVQ287, produces lipochitooligosaccharide Nod factors (LCOs) that differ from those of the parental strain. The nonreducing N-acetylglucosamine residues of all of the LCOs of mutant SVQ287 lack fucose and 2-O-methylfucose substituents. In addition, SVQ287 synthesizes an LCO with an unusually long, C20:1 fatty acyl side chain. The transposon insertion of mutant SVQ287 lies within a 1.1-kb HindIII fragment. This and an adjacent 2.4-kb HindIII fragment were sequenced. The sequence contains the 3' end of noeK, nodZ, and noeL (the gene interrupted by Tn5-B20), and the 5' end of nolK, all in the same orientation. Although each of these genes has a similarly oriented counterpart on the symbiosis plasmid of the broad-host-range Rhizobium sp. strain NGR234, there are significant differences in the noeK/nodZ intergenic region. Based on amino acid sequence homology, noeL encodes GDP-D-mannose dehydratase, an enzyme involved in the synthesis of GDP-L-fucose, and nolK encodes a NAD-dependent nucleotide sugar epimerase/dehydrogenase. We show that expression of the noeL gene is under the control of NodD1 in S. fredii and is most probably mediated by the nod box that precedes nodZ. Transposon insertion into neoL has two impacts on symbiosis with Williams soybean: nodulation rate is reduced slightly and competitiveness for nodulation is decreased significantly. Mutant SVQ287 retains its ability to form nitrogen-fixing nodules on other legumes, but final nodule number is attenuated on Cajanus cajan.     

Stereochemistry and mechanism of the GDP-mannose dehydratase reaction.

The reaction catalyzed by bacterial GDP-mannose dehydratase (E.C. 4.2.1.47), the conversion of GDP-D-mannose to GDP-4-keto-6-deoxymannose (GDP-6-deoxy-D-lyxo-hexos-4-ulose), was studied with (6R)- and (6S)-GDP-D-[4-2H1,6-3H]mannose. Conversion of these stereospecifically labeled substrates in the presence of excess unlabeled GDP-mannose into the 4-keto-6-deoxy derivatives followed by Kuhn-Roth oxidation gave acetic acid samples which were subjected to configurational analysis of the isotopically chiral methyl group. The observed F values of 64 for the material from the (6S) substrate and 31 for that from the (6R) isomer, corresponding to 48% e.e. R and 66% e.e. S configuration, respectively, of the methyl group indicate that (a) the oxidoreductase reaction involves transfer of H-4 to C-6, (b) the transfer is predominantly intramolecular, and (c) the transfer is stereospecific, H-4 replacing the C-6 hydroxyl group with inversion of configuration. A mechanism for the reaction is proposed on the basis of these results.