Structure, mechanism and engineering of a nucleotidylyltransferase as a first step toward glycorandomization

Metabolite glycosylation is affected by three classes of enzymes: nucleotid ylyltransferases, which activate sugars as nucleotide diphospho-derivatives , intermediate sugar-modifying enzymes and glycosyltransferases. which tran sfer the final derivatized activated sugars to aglycon substrates. One of t he first crystal structures of an enzyme responsible for the first step in this cascade, alpha -D-glucopyranosyl phosphate thymidylyltransferase (E-p) from Salmonella, in complex with product (UDP-Glc) and substrate (dTTP) is reported at 2.0 Angstrom and 2.1 Angstrom resolution, respectively. These structures, in conjunction with the kinetic characterization of E-p, clarif y the catalytic mechanism of this important enzyme class. Structure-based e ngineering of E-p produced modified enzymes capable of utilizing 'unnatural ' sugar phosphates not accepted by wild type E-p. The demonstrated ability to alter nucleotidylyltransferase specificity by design is an integral comp onent of in vitro glycosylation systems developed for the production of div erse glycorandomized libraries.