The donor subsite of trehalose-6-phosphate synthase - Binary complexes with UDP-glucose and UDP-2-deoxy-2-fluoro-glucose at 2 angstrom resolution

R P  Gibson; C A  Tarling; S  Roberts; S G  Withers; G J  Davies

doi:10.1074/jbc.M307643200

The donor subsite of trehalose-6-phosphate synthase - Binary complexes with UDP-glucose and UDP-2-deoxy-2-fluoro-glucose at 2 angstrom resolution

R P Gibson, C A Tarling, S Roberts, S G Withers, G J Davies

Chemistry

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

Abstract

Trehalose is an unusual non-reducing disaccharide that plays a variety of biological roles, from food storage to cellular protection from environmental stresses such as desiccation, pressure, heat-shock, extreme cold, and oxygen radicals. It is also an integral component of the cell-wall glycolipids of mycobacteria. The primary enzymatic route to trehalose first involves the transfer of glucose from a UDP-glucose donor to glucose-6-phosphate to form alpha,alpha-1,1 trehalose-6-phosphate. This reaction, in which the configurations of two glycosidic bonds are set simultaneously, is catalyzed by the glycosyltransferase trehalose-6-phosphate synthase ( OtsA), which acts with retention of the anomeric configuration of the UDP-sugar donor. The classification of activated sugar-dependent glycosyltransferases into approximately 70 distinct families based upon amino acid sequence similarities places OtsA in glycosyltransferase family 20 (see afmb.cnrs-mrs.fr/CAZY/). The recent 2.4 Angstrom structure of Escherichia coli OtsA revealed a two-domain enzyme with catalysis occurring at the interface of the twin beta/alpha/beta domains. Here we present the 2.0 Angstrom structures of the E. coli OtsA in complex with either UDP-Glc or the non-transferable analogue UDP-2-deoxy-2-fluoroglucose. Both complexes unveil the donor subsite interactions, confirming a strong similarity to glycogen phosphorylases, and reveal substantial conformational differences to the previously reported complex with UDP and glucose 6-phosphate. Both the relative orientation of the two domains and substantial ( up to 10 Angstrom) movements of an N-terminal loop ( residues 9 - 22) characterize the more open "relaxed" conformation of the binary UDP-sugar complexes reported here.

Original language	English
Pages (from-to)	1950-1955
Number of pages	6
Journal	Journal of Biological Chemistry
Volume	279
Issue number	3
DOIs	https://doi.org/10.1074/jbc.M307643200
Publication status	Published - 16 Jan 2004

Keywords

CRYSTAL-STRUCTURE
CATALYTIC MECHANISM
ESCHERICHIA-COLI
CORYNEBACTERIUM-GLUTAMICUM
SACCHAROMYCES-CEREVISIAE
BETA-GLUCOSYLTRANSFERASE
GLYCOGEN-PHOSPHORYLASE
NEISSERIA-MENINGITIDIS
TREHALOSE ACCUMULATION
GLYCOSIDE HYDROLASES

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10.1074/jbc.M307643200

Cite this

@article{c0506f5a34e64fe98a34b96709cc88f2,

title = "The donor subsite of trehalose-6-phosphate synthase - Binary complexes with UDP-glucose and UDP-2-deoxy-2-fluoro-glucose at 2 angstrom resolution",

abstract = "Trehalose is an unusual non-reducing disaccharide that plays a variety of biological roles, from food storage to cellular protection from environmental stresses such as desiccation, pressure, heat-shock, extreme cold, and oxygen radicals. It is also an integral component of the cell-wall glycolipids of mycobacteria. The primary enzymatic route to trehalose first involves the transfer of glucose from a UDP-glucose donor to glucose-6-phosphate to form alpha,alpha-1,1 trehalose-6-phosphate. This reaction, in which the configurations of two glycosidic bonds are set simultaneously, is catalyzed by the glycosyltransferase trehalose-6-phosphate synthase ( OtsA), which acts with retention of the anomeric configuration of the UDP-sugar donor. The classification of activated sugar-dependent glycosyltransferases into approximately 70 distinct families based upon amino acid sequence similarities places OtsA in glycosyltransferase family 20 (see afmb.cnrs-mrs.fr/CAZY/). The recent 2.4 Angstrom structure of Escherichia coli OtsA revealed a two-domain enzyme with catalysis occurring at the interface of the twin beta/alpha/beta domains. Here we present the 2.0 Angstrom structures of the E. coli OtsA in complex with either UDP-Glc or the non-transferable analogue UDP-2-deoxy-2-fluoroglucose. Both complexes unveil the donor subsite interactions, confirming a strong similarity to glycogen phosphorylases, and reveal substantial conformational differences to the previously reported complex with UDP and glucose 6-phosphate. Both the relative orientation of the two domains and substantial ( up to 10 Angstrom) movements of an N-terminal loop ( residues 9 - 22) characterize the more open {"}relaxed{"} conformation of the binary UDP-sugar complexes reported here.",

keywords = "CRYSTAL-STRUCTURE, CATALYTIC MECHANISM, ESCHERICHIA-COLI, CORYNEBACTERIUM-GLUTAMICUM, SACCHAROMYCES-CEREVISIAE, BETA-GLUCOSYLTRANSFERASE, GLYCOGEN-PHOSPHORYLASE, NEISSERIA-MENINGITIDIS, TREHALOSE ACCUMULATION, GLYCOSIDE HYDROLASES",

author = "Gibson, {R P} and Tarling, {C A} and S Roberts and Withers, {S G} and Davies, {G J}",

year = "2004",

month = jan,

day = "16",

doi = "10.1074/jbc.M307643200",

language = "English",

volume = "279",

pages = "1950--1955",

journal = "Journal of Biological Chemistry",

issn = "0021-9258",

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

number = "3",

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T1 - The donor subsite of trehalose-6-phosphate synthase - Binary complexes with UDP-glucose and UDP-2-deoxy-2-fluoro-glucose at 2 angstrom resolution

AU - Gibson, R P

AU - Tarling, C A

AU - Roberts, S

AU - Withers, S G

AU - Davies, G J

PY - 2004/1/16

Y1 - 2004/1/16

N2 - Trehalose is an unusual non-reducing disaccharide that plays a variety of biological roles, from food storage to cellular protection from environmental stresses such as desiccation, pressure, heat-shock, extreme cold, and oxygen radicals. It is also an integral component of the cell-wall glycolipids of mycobacteria. The primary enzymatic route to trehalose first involves the transfer of glucose from a UDP-glucose donor to glucose-6-phosphate to form alpha,alpha-1,1 trehalose-6-phosphate. This reaction, in which the configurations of two glycosidic bonds are set simultaneously, is catalyzed by the glycosyltransferase trehalose-6-phosphate synthase ( OtsA), which acts with retention of the anomeric configuration of the UDP-sugar donor. The classification of activated sugar-dependent glycosyltransferases into approximately 70 distinct families based upon amino acid sequence similarities places OtsA in glycosyltransferase family 20 (see afmb.cnrs-mrs.fr/CAZY/). The recent 2.4 Angstrom structure of Escherichia coli OtsA revealed a two-domain enzyme with catalysis occurring at the interface of the twin beta/alpha/beta domains. Here we present the 2.0 Angstrom structures of the E. coli OtsA in complex with either UDP-Glc or the non-transferable analogue UDP-2-deoxy-2-fluoroglucose. Both complexes unveil the donor subsite interactions, confirming a strong similarity to glycogen phosphorylases, and reveal substantial conformational differences to the previously reported complex with UDP and glucose 6-phosphate. Both the relative orientation of the two domains and substantial ( up to 10 Angstrom) movements of an N-terminal loop ( residues 9 - 22) characterize the more open "relaxed" conformation of the binary UDP-sugar complexes reported here.

AB - Trehalose is an unusual non-reducing disaccharide that plays a variety of biological roles, from food storage to cellular protection from environmental stresses such as desiccation, pressure, heat-shock, extreme cold, and oxygen radicals. It is also an integral component of the cell-wall glycolipids of mycobacteria. The primary enzymatic route to trehalose first involves the transfer of glucose from a UDP-glucose donor to glucose-6-phosphate to form alpha,alpha-1,1 trehalose-6-phosphate. This reaction, in which the configurations of two glycosidic bonds are set simultaneously, is catalyzed by the glycosyltransferase trehalose-6-phosphate synthase ( OtsA), which acts with retention of the anomeric configuration of the UDP-sugar donor. The classification of activated sugar-dependent glycosyltransferases into approximately 70 distinct families based upon amino acid sequence similarities places OtsA in glycosyltransferase family 20 (see afmb.cnrs-mrs.fr/CAZY/). The recent 2.4 Angstrom structure of Escherichia coli OtsA revealed a two-domain enzyme with catalysis occurring at the interface of the twin beta/alpha/beta domains. Here we present the 2.0 Angstrom structures of the E. coli OtsA in complex with either UDP-Glc or the non-transferable analogue UDP-2-deoxy-2-fluoroglucose. Both complexes unveil the donor subsite interactions, confirming a strong similarity to glycogen phosphorylases, and reveal substantial conformational differences to the previously reported complex with UDP and glucose 6-phosphate. Both the relative orientation of the two domains and substantial ( up to 10 Angstrom) movements of an N-terminal loop ( residues 9 - 22) characterize the more open "relaxed" conformation of the binary UDP-sugar complexes reported here.

KW - CRYSTAL-STRUCTURE

KW - CATALYTIC MECHANISM

KW - ESCHERICHIA-COLI

KW - CORYNEBACTERIUM-GLUTAMICUM

KW - SACCHAROMYCES-CEREVISIAE

KW - BETA-GLUCOSYLTRANSFERASE

KW - GLYCOGEN-PHOSPHORYLASE

KW - NEISSERIA-MENINGITIDIS

KW - TREHALOSE ACCUMULATION

KW - GLYCOSIDE HYDROLASES

U2 - 10.1074/jbc.M307643200

DO - 10.1074/jbc.M307643200

M3 - Article

SN - 0021-9258

VL - 279

SP - 1950

EP - 1955

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 3

ER -