Substrate and Metal Ion Promiscuity in Mannosylglycerate Synthase

Morten M. Nielsen; Michael D. L. Suits; Min Yang; Conor S. Barry; Carlos Martinez-Fleites; Louise E. Tailford; James E. Flint; Claire Dumon; Benjamin G. Davis; Harry J. Gilbert; Gideon J. Davies

doi:10.1074/jbc.M110.199844

Substrate and Metal Ion Promiscuity in Mannosylglycerate Synthase

Morten M. Nielsen, Michael D. L. Suits, Min Yang, Conor S. Barry, Carlos Martinez-Fleites, Louise E. Tailford, James E. Flint, Claire Dumon, Benjamin G. Davis, Harry J. Gilbert^*, Gideon J. Davies

^*Corresponding author for this work

Chemistry

Research output: Contribution to journal › Article › peer-review

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 alpha-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.

Original language	English
Pages (from-to)	15155-15164
Number of pages	10
Journal	Journal of Biological Chemistry
Volume	286
Issue number	17
DOIs	https://doi.org/10.1074/jbc.M110.199844
Publication status	Published - 29 Apr 2011

Keywords

ALPHA-1,3-GALACTOSYLTRANSFERASE
COMPLEX
BLOOD-GROUP
ENZYMES
DONOR
GLYCOSYLTRANSFERASES
INSIGHT
DIFFRACTION DATA
CRYSTAL-STRUCTURE
MECHANISMS

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10.1074/jbc.M110.199844

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title = "Substrate and Metal Ion Promiscuity in Mannosylglycerate Synthase",

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 alpha-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.",

keywords = "ALPHA-1,3-GALACTOSYLTRANSFERASE, COMPLEX, BLOOD-GROUP, ENZYMES, DONOR, GLYCOSYLTRANSFERASES, INSIGHT, DIFFRACTION DATA, CRYSTAL-STRUCTURE, MECHANISMS",

author = "Nielsen, {Morten M.} and Suits, {Michael D. L.} and Min Yang and Barry, {Conor S.} and Carlos Martinez-Fleites and Tailford, {Louise E.} and Flint, {James E.} and Claire Dumon and Davis, {Benjamin G.} and Gilbert, {Harry J.} and Davies, {Gideon J.}",

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day = "29",

doi = "10.1074/jbc.M110.199844",

language = "English",

volume = "286",

pages = "15155--15164",

journal = "Journal of Biological Chemistry",

issn = "0021-9258",

publisher = "American Society for Biochemistry and Molecular Biology Inc.",

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T1 - Substrate and Metal Ion Promiscuity in Mannosylglycerate Synthase

AU - Nielsen, Morten M.

AU - Suits, Michael D. L.

AU - Yang, Min

AU - Barry, Conor S.

AU - Martinez-Fleites, Carlos

AU - Tailford, Louise E.

AU - Flint, James E.

AU - Dumon, Claire

AU - Davis, Benjamin G.

AU - Gilbert, Harry J.

AU - Davies, Gideon J.

PY - 2011/4/29

Y1 - 2011/4/29

N2 - 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 alpha-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.

AB - 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 alpha-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.

KW - ALPHA-1,3-GALACTOSYLTRANSFERASE

KW - COMPLEX

KW - BLOOD-GROUP

KW - ENZYMES

KW - DONOR

KW - GLYCOSYLTRANSFERASES

KW - INSIGHT

KW - DIFFRACTION DATA

KW - CRYSTAL-STRUCTURE

KW - MECHANISMS

UR - http://www.scopus.com/inward/record.url?scp=79955426783&partnerID=8YFLogxK

U2 - 10.1074/jbc.M110.199844

DO - 10.1074/jbc.M110.199844

M3 - Article

SN - 0021-9258

VL - 286

SP - 15155

EP - 15164

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 17

ER -

Substrate and Metal Ion Promiscuity in Mannosylglycerate Synthase

Abstract

Keywords

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