Substrate and metal ion promiscuity in mannosylglycerate synthase

Nielsen, Morten M. and Suits, Michael D. L. and Yang, Min and Barry, Conor S. and Martinez-Fleites, Carlos and Tailford, Louise E. and Flint, James E. and Dumon, Claire and Davis, Benjamin G. and Gilbert, Harry J. and Davies, Gideon J. (2011) Substrate and metal ion promiscuity in mannosylglycerate synthase. Journal of Biological Chemistry, 286 (17). pp. 15155-15164. ISSN 0021-9258

Full content URL: http://dx.doi.org/10.1074/jbc.M110.199844

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Item Type:Article
Item Status:Live Archive

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 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 iondependent 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. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Additional Information:First Published [online] on February 2, 2011
Keywords:Active center, Anomeric configuration, Calorimetric analysis, Catalytic mechanisms, Cellular biology, Divalent metal ion, Glycoconjugates, Glycosyl, Glycosyl transferase, Ion-dependence, Key component, Key factors, Mannosylglycerate, Metal binding, Metal coordination, Metal specificity, Metal-coordination sites, Model system, Substrate binding, Synthases, Wild types, Catalysis, Coordination reactions, Cytology, Manganese, Metal ions, Sugars, Synthesis (chemical), Metals, calcium ion, cobalt, glyceric acid, guanine, histidine, lactic acid, magnesium ion, mannosylglycerate synthase, metal ion, nickel, synthetase, tyrosine, unclassified drug, amino acid substitution, article, binding kinetics, calorimetry, catalysis, conformation, controlled study, enzyme analysis, enzyme structure, molecular cloning, nonhuman, priority journal, protein expression, Rhodothermus, Rhodothermus marinus, site directed mutagenesis, wild type, Bacterial Proteins, Calcium, Kinetics, Magnesium, Mannosyltransferases, Metals, Mutagenesis, Site-Directed, Substrate Specificity, Rhodothermus marinus
Subjects:C Biological Sciences > C720 Biological Chemistry
Divisions:College of Science > School of Life Sciences
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ID Code:12456
Deposited On:16 Dec 2013 15:15

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