The Donor Subsite of Trehalose-6-phosphate Synthase

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 α,α-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 Å structure of Escherichia coli OtsA revealed a two-domain enzyme with catalysis occurring at the interface of the twin β/α/β domains. Here we present the 2.0 Å 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 Å) movements of an N-terminal loop (residues 9–22) characterize the more open “relaxed” conformation of the binary UDP-sugar complexes reported here.