Glycosyltransferases involved in biosynthesis of the outer core region of Escherichia coli lipopolysaccharides exhibit broader substrate specificities than is predicted from lipopolysaccharide structures

Abstract

The waaJ, waaT, and waaR genes encode α-1,2-glycosyltransferases involved in synthesis of the outer core region of the lipopolysaccharide of Escherichia coli. They belong to the glycosyltransferase CAZy family 8, characterized by the GT-A fold, DXD motifs, and by retention of configuration at the anomeric carbon of the donor sugar. Each enzyme adds a hexose residue at the same stage of core oligosaccharide backbone extension. However, they differ in the epimers for their donor nucleotide sugars, and in their acceptor residues. WaaJ is a UDP-glucose: (galactosyl) LPS α-1,2- glucosyltransferase, whereas WaaR and WaaT have UDP-glucose:(glucosyl) LPS α-1,2-glucosyltransferase and UDP-galactose:(glucosyl) LPS α-1,2-galactosyltransferase activities, respectively. The objective of this work was to examine their ability to utilize alternate donors and acceptors. When expressed in the heterologous host, each enzyme was able to extend the alternate LPS acceptor in vivo but they retained their natural donor specificity. In vitro assays were then performed to test the effect of substituting the epimeric donor sugar on incorporation efficiency with the natural LPS acceptor of the enzyme. Although each enzyme could utilize the alternate donor epimer, activity was compromised because of significant decreases in kcat and corresponding increases in K m(donor). Finally, in vitro assays were performed to probe acceptor preference in the absence of the cellular machinery. The results were enzyme-dependent: while an alternate acceptor had no significant effect on the kinetic behavior of His6-WaaT, His6-WaaJ showed a significantly decreased kcat and increased Km(acceptor). These results illustrate the differences in behavior between closely related glycosyltransferase enzymes involved in the synthesis of similar glycoconjugates and have implications for glycoengineering applications. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.