Kinetic and crystallographic analyses support a sequential-ordered BiBi catalytic mechanism for Escherichia coli glucose-1-phosphate thymidylyltransferase

Glucose-1-phosphate thymidylyltransferase is the first enzyme in the biosyn thesis of dTDP-L-rhamnose, the precursor Of L-rhamnose, an essential compon ent of surface antigens, such as the O-lipopolysaccharide, mediating virule nce and adhesion to host tissues in many microorganisms. The enzyme catalys es the formation of dTDP-glucose, from dTTP and glucose 1-phosphate, as wel l as its pyrophosphorolysis. To shed more light on the catalytic properties of glucose-1-phosphate thymidylyltransferase from Escherichia coli, specif ically distinguishing between ping pong and sequential ordered bi bi reacti on mechanisms, the enzyme kinetic properties have been analysed in the pres ence of different substrates and inhibitors. Moreover, three different comp lexes of glucose-1-phosphate thymidylyltransferase (co-crystallized with dT DP, with dTMP and glucose-l-phosphate, with D-thymidine and glucose-l-phosp hate) have been analysed by X-ray crystallography, in the 1.9-2.3 Angstrom resolution range (R-factors, of 17.3-17.5%). The homotetrameric enzyme show s strongly conserved substrate/inhibitor binding modes in a surface cavity next to the topological switch-point of a quasi-Rossmann fold. Inspection o f the subunit tertiary structure reveals relationships to other enzymes inv olved in the biosynthesis of nucleotide-sugars, including distant proteins such as the molybdenum cofactor biosynthesis protein MobA. The precise loca tion of the substrate relative to putative reactive residues in the catalyt ic center suggests that, in keeping with the results of the kinetic measure ments, both catalysed reactions, i.e. dTDP-glucose biosynthesis and pyropho sphorolysis, follow a sequential ordered bi bi catalytic mechanism. (C) 200 1 Academic Press.