The donor subsite of trehalose-6-phosphate synthase: binary complexes with UDP-glucose and UDP-2-deoxy-2-fluoro-glucose at 2 A resolution.
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 A 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 A 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 A) movements of an N-terminal loop (residues 9-22) characterize the more open "relaxed" conformation of the binary UDP-sugar complexes reported here.
Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5YW, United Kingdom.
Robert P
RP
Gibson
Chris A
CA
Tarling
Shirley
S
Roberts
Stephen G
SG
Withers
Gideon J
GJ
Davies
D002384:Catalysis; D003847:Deoxyglucose / Q000031:analogs & derivatives* / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005964:Glucosyltransferases / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D014532:Uridine Diphosphate Glucose / Q000737:chemistry*
D029968:Escherichia coli Proteins; C041530:uridine-2-deoxy-2-fluoro-D-glucose diphosphate ester; D003847:Deoxyglucose; D005964:Glucosyltransferases; C023180:trehalose-6-phosphate synthase; D014532:Uridine Diphosphate Glucose