title: Biochemical characterization of the retaining glycosyltransferase glucosyl-3-phosphoglycerate synthase from Mycobacterium tuberculosis.
abstract: Glucosyl-3-phosphoglycerate synthase (GpgS) catalyzes the first step in the biosynthesis of glucosyl glycerate, the putative precursor used in building methylated polysaccharides in mycobacteria. Enzymes from Mycobacterium tuberculosis (MtGpgS) and related species have been structurally characterized and subjected to basic kinetic analyses, but more in-depth kinetic analysis is currently lacking. Dead-end inhibition studies with MtGpgS suggest an ordered kinetic mechanism with 3-phosphoglycerate (3-PGA) binding first, followed by UDP-glucose, in contrast to previous reports. At higher concentrations, 3-PGA exhibits competitive substrate inhibition vs. UDP-glucose, suggesting 3-PGA can bind to either binding site on the enzyme. Parabolic noncompetitive inhibition plots by a 3-PGA analog also support this conclusion. The effect of varying pH on the catalytic parameters indicates single ionizable residue involved catalysis (pKa=6.3) that must be deprotonated for full activity. A solvent kinetic isotope effect of 2.0±0.3 on kcat is consistent with a proton in flight during the rate-determining step. Site-directed mutagenesis studies identify several residues critical for interactions with substrates. Although the residues are conserved among other glycosyltransferase families catalyzing similar reactions, the effect of substitutions varies between families suggesting that conserved areas play different catalytic roles in each family.
authors.affiliation: Department of Chemistry, The University of Alabama, Box 870336, Tuscaloosa, AL 35487, USA.
authors.forename: Garima
authors.initials: G
authors.lastname: Kumar
authors.affiliation: Department of Chemistry, The University of Alabama, Box 870336, Tuscaloosa, AL 35487, USA.
authors.forename: Siqi
authors.initials: S
authors.lastname: Guan
authors.affiliation: Department of Chemistry, The University of Alabama, Box 870336, Tuscaloosa, AL 35487, USA. Electronic address: pfrantom@ua.edu.
authors.forename: Patrick A
authors.initials: PA
authors.lastname: Frantom
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005988:Glyceric Acids / Q000737:chemistry* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D011485:Protein Binding; D014532:Uridine Diphosphate Glucose / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005988:Glyceric Acids; C005156:3-phosphoglycerate; D005964:Glucosyltransferases; C534794:glucosyl-3-phosphoglycerate synthase, Mycobacterium tuberculosis; D014532:Uridine Diphosphate Glucose
keywords: GT-A fold; Glucosyl-3-phosphoglycerate synthase; Kinetic mechanism; Methylated polysaccharides; Retaining glycosyltransferase; Substrate inhibition