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I03-Macromolecular Crystallography	Substrate and metal-ion promiscuity in mannosylglycerate synthase Morten Nielsen , Michael Suits , Min Yang , Conor Barry , Carlos Martinez-Fleites , Louise Tailford , James Flint , Claire Dumon , Benjamin Davis , Harry Gilbert , Gideon Davies Journal Of Biological Chemistry DOI: 10.1074/jbc.M110.199844 PMID: 21288903 Open Access Show Abstract ▼ Abstract: 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 glycosyltransfer. Mannosylglycerate synthase (MGS) catalyzes the synthesis of a-mannosyl-D-glycerate using GDP-mannose as the preferred donor species; a reaction that occurs with 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 Mg2+, Ca2+and, to a varying extent, Mn2+, Ni2+, and Co2+, 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 kcat whilst substitution of H217, a key component of the metal-coordination site, results in a change in metal specificity to Mn2+. Structural analyses of MGS complexes not only provide insight into metal coordination but also into how lactate can function as an alternative acceptor to glycerate. These studies highlight the role of flexible loops in the active centre and the subsequent coordination of the divalent metal-ion as key factors in MGS catalysis and metal-ion dependence. Furthermore, Y220, located on a flexible loop whose conformation is likely influenced by metal binding also plays a critical role in substrate binding.	Feb 2011

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I03-Macromolecular Crystallography

Substrate and metal-ion promiscuity in mannosylglycerate synthase