Publications

• Biosynthesis of mannosylglycerate in the thermophilic bacterium Rhodothermus marinus. Biochemical and genetic characterization of a mannosylglycerate synthase.

  Martins L.O., Empadinhas N., Marugg J.D., Miguel C., Ferreira C., da Costa M.S., Santos H.

  The biosynthetic reaction scheme for the compatible solute mannosylglycerate in Rhodothermus marinus is proposed based on measurements of the relevant enzymatic activities in cell-free extracts and in vivo (13)C labeling experiments. The synthesis of mannosylglycerate proceeded via two alternative ... >> More

  The biosynthetic reaction scheme for the compatible solute mannosylglycerate in Rhodothermus marinus is proposed based on measurements of the relevant enzymatic activities in cell-free extracts and in vivo (13)C labeling experiments. The synthesis of mannosylglycerate proceeded via two alternative pathways; in one of them, GDP mannose was condensed with D-glycerate to produce mannosylglycerate in a single reaction catalyzed by mannosylglycerate synthase, in the other pathway, a mannosyl-3-phosphoglycerate synthase catalyzed the conversion of GDP mannose and D-3-phosphoglycerate into a phosphorylated intermediate, which was subsequently converted to mannosylglycerate by the action of a phosphatase. The enzyme activities committed to the synthesis of mannosylglycerate were not influenced by the NaCl concentration in the growth medium. However, the combined mannosyl-3-phosphoglycerate synthase/phosphatase system required the addition of NaCl or KCl to the assay mixture for optimal activity. The mannosylglycerate synthase enzyme was purified and characterized. Based on partial sequence information, the corresponding mgs gene was identified from a genomic library of R. marinus. In addition, the mgs gene was overexpressed in Escherichia coli with a high yield. The enzyme had a molecular mass of 46,125 Da, and was specific for GDP mannose and D-glycerate. This is the first report of the characterization of a mannosylglycerate synthase. << Less

  J. Biol. Chem. 274:35407-35414(1999) [PubMed] [EuropePMC]

• Substrate and metal ion promiscuity in mannosylglycerate synthase.

  Nielsen M.M., Suits M.D., Yang M., Barry C.S., Martinez-Fleites C., Tailford L.E., Flint J.E., Dumon C., Davis B.G., Gilbert H.J., Davies G.J.

  The enzymatic transfer of the sugar mannose from activated sugar donors is central to the synthesis of a wide range of biologically significant polysaccharides and glycoconjugates. In addition to their importance in cellular biology, mannosyltransferases also provide model systems with which to st ... >> More

  The enzymatic transfer of the sugar mannose from activated sugar donors is central to the synthesis of a wide range of biologically significant polysaccharides and glycoconjugates. In addition to their importance in cellular biology, mannosyltransferases also provide model systems with which to study catalytic mechanisms of glycosyl transfer. Mannosylglycerate synthase (MGS) catalyzes the synthesis of α-mannosyl-D-glycerate using GDP-mannose as the preferred donor species, a reaction that occurs with a net retention of anomeric configuration. Past work has shown that the Rhodothermus marinus MGS, classified as a GT78 glycosyltransferase, displays a GT-A fold and performs catalysis in a metal ion-dependent manner. MGS shows very unusual metal ion dependences with Mg(2+) and Ca(2+) and, to a lesser extent, Mn(2+), Ni(2+), and Co(2+), thus facilitating catalysis. Here, we probe these dependences through kinetic and calorimetric analyses of wild-type and site-directed variants of the enzyme. Mutation of residues that interact with the guanine base of GDP are correlated with a higher k(cat) value, whereas substitution of His-217, a key component of the metal coordination site, results in a change in metal specificity to Mn(2+). Structural analyses of MGS complexes not only provide insight into metal coordination but also how lactate can function as an alternative acceptor to glycerate. These studies highlight the role of flexible loops in the active center and the subsequent coordination of the divalent metal ion as key factors in MGS catalysis and metal ion dependence. Furthermore, Tyr-220, located on a flexible loop whose conformation is likely influenced by metal binding, also plays a critical role in substrate binding. << Less

  J. Biol. Chem. 286:15155-15164(2011) [PubMed] [EuropePMC]

• Structural dissection and high-throughput screening of mannosylglycerate synthase.

  Flint J., Taylor E., Yang M., Bolam D.N., Tailford L.E., Martinez-Fleites C., Dodson E.J., Davis B.G., Gilbert H.J., Davies G.J.

  The enzymatic transfer of activated mannose yields mannosides in glycoconjugates and oligo- and polysaccharides. Yet, despite its biological necessity, the mechanism by which glycosyltransferases recognize mannose and catalyze its transfer to acceptor molecules is poorly understood. Here, we repor ... >> More

  The enzymatic transfer of activated mannose yields mannosides in glycoconjugates and oligo- and polysaccharides. Yet, despite its biological necessity, the mechanism by which glycosyltransferases recognize mannose and catalyze its transfer to acceptor molecules is poorly understood. Here, we report broad high-throughput screening and kinetic analyses of both natural and synthetic substrates of Rhodothermus marinus mannosylglycerate synthase (MGS), which catalyzes the formation of the stress protectant 2-O-alpha-D-mannosyl glycerate. The sequence of MGS indicates that it is at the cusp of inverting and retaining transferases. The structures of apo MGS and complexes with donor and acceptor molecules, including GDP-mannose, combined with mutagenesis of the binding and catalytic sites, unveil the mannosyl transfer center. Nucleotide specificity is as important in GDP-D-mannose recognition as the nature of the donor sugar. << Less

  Nat. Struct. Mol. Biol. 12:608-614(2005) [PubMed] [EuropePMC]

• The plant Selaginella moellendorffii possesses enzymes for synthesis and hydrolysis of the compatible solutes mannosylglycerate and glucosylglycerate.

  Nobre A., Empadinhas N., Nobre M.F., Lourenco E.C., Maycock C., Ventura M.R., Mingote A., da Costa M.S.

  A mannosylglycerate synthase (MgS) gene detected in the genome of Selaginella moellendorffii was expressed in E. coli and the recombinant enzyme was purified and characterized. A remarkable and unprecedented feature of this enzyme was the ability to efficiently synthesize mannosylglycerate (MG) an ... >> More

  A mannosylglycerate synthase (MgS) gene detected in the genome of Selaginella moellendorffii was expressed in E. coli and the recombinant enzyme was purified and characterized. A remarkable and unprecedented feature of this enzyme was the ability to efficiently synthesize mannosylglycerate (MG) and glucosylglycerate (GG) alike, with maximal activity at 50 °C, pH 8.0 and with Mg(2+) as reaction enhancer. We have also identified a novel glycoside hydrolase gene in this plant's genome, which was functionally confirmed to be highly specific for the hydrolysis of MG and GG and named MG hydrolase (MgH), due to its homology with bacterial MgHs. The recombinant enzyme was maximally active at 40 °C and at pH 6.0-6.5. The activity was independent of cations, but Mn(2+) was a strong stimulator. Regardless of these efficient enzymatic resources we could not detect MG or GG in S. moellendorffii or in the extracts of five additional Selaginella species. Herein, we describe the properties of the first eukaryotic enzymes for the synthesis and hydrolysis of the compatible solutes, MG and GG. << Less

  Planta 237:891-901(2013) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.