Two sequence elements of glycosyltransferases involved in urdamycin biosynthesis are responsible for substrate specificity and enzymatic activity

Background: Two deoxysugar glycosyltransferases (GTs), UrdGT1b and UrdGT1c, involved in urdamycin biosynthesis share 91% identical amino acids. Howeve r, the two GTs show different specificities for both nucleotide sugar and a cceptor substrate. Generally, it is proposed that GTs are two-domain protei ns with a nucleotide binding domain and an acceptor substrate site with the catalytic center in an interface cleft between these domains. Our work aim ed at finding out the region responsible for determination of substrate spe cificities of these two urdamycin GTs. Results: A series of 10 chimeric GT genes were constructed consisting of di fferently sized and positioned portions of urdGT1b and urdCT1c. Gene expres sion experiments in host strains Streptomyces fradiae Ax and XTC show that nine of 10 chimeric GTs are still functional, with either UrdGT1b- or UrdGT 1c-like activity. A 31 amino acid region (aa 52-82) located close to the N- terminus of these enzymes, which differs in 18 residues, was identified to control both sugar donor and acceptor substrate specificity. Only one chime ric gene product of the 10 was not functional. Targeted stepwise alteration s of glycine 226 (G226R, G226S, G226SR) were made to reintroduce residues c onserved among streptomycete GTs. Alterations G226S and G226R restored a we ak activity, whereas G226SR showed an activity comparable with other functi onal chimeras. Conclusions: A nucleotide sugar binding motif is present in the C-terminal moiety of UrdGT1b and UrdGT1c from S. fradiae. We could demonstrate that it is an N-terminal section that determines specificity for the nucleotide su gar and also the acceptor substrate. This finding directs the way towards e ngineering this class of streptomycete enzymes for antibiotic derivatizatio n applications. Amino acids 226 and 227, located outside the putative subst rate binding site, might be part of a larger protein structure, perhaps a s olvent channel to the catalytic center. Therefore, they could play a role i n substrate accessibility to it. (C) 2001 Elsevier Science Ltd. All rights reserved.