Mechanistic Studies and Catalytic Engineering of the Pseudoglycosyltransferase VldE

Many bioactive natural products contain sugar moieties in their structures. These sugars may play an important role in their biological activities. The attachment of the sugars to the core natural product structures is catalyzed by the glycosyltransferases (GTs). Currently, there are more than 140,000 putative GTs listed in the CAZY database. However, only a portion of them has been carefully characterized. During our study on the biosynthesis of the antifungal antibiotic validamycin A, we discovered a glycosyltransferase-like protein (VldE) that is similar to trehalose 6-phosphate synthase (OtsA). However, in contrast to OtsA, which catalyzes a coupling reaction between UDP-glucose and glucose 6-phosphate, VldE catalyzes a coupling reaction between two non-sugar substrates, GDP-valienol and validamine 7’-phosphate, to give validoxylamine A 7’-phosphate as product. It was unclear how a glycosyltransferase-like enzyme could catalyze a coupling reaction between two non-sugar molecules. To understand the catalytic mechanism of this unusual enzyme we performed a comparative study and protein engineering by swapping the N- and C-terminal domains of VldE with those of OtsA from Streptomyces coelicolor. The resulting chimeric proteins were tested using a variety of substrates. The results provide insights into the unique catalytic mechanism of pseudoglycosyltransferases. The knowledge may also be used to develop new tools for generating novel glycosylated natural products and redesigning glycoconjugates.