A kinetic characterization of the glycosyltransferase activity of Eschericia coli PBP1b and development of a continuous fluorescence assay.
The bacterial cell wall is a polymer consisting of alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) units, cross-linked via peptides appended to MurNAc. The final steps in the formation of cell wall, also referred to as murein, are catalyzed by high-molecular-weight, class A penicillin-binding proteins (PBPs). These bifunctional enzymes catalyze both glycosyltransfer, to form the carbohydrate backbone of murein, and transpeptidation, to form the interstrand peptide linkages. Using PBP1b from Eschericia coli, an in vitro kinetic characterization of the glycosyltransfer reaction was carried out. Initial studies with unlabeled substrate (Lipid II) revealed that activity is strongly influenced by DMSO, as well as metal and detergent. In addition, a continuous fluoresence assay was developed and used to determine the effect of pH on the reaction. A single basic residue was titrated, with a pK(a) of 7.0. Taken together, these data suggest a mechanism for PBP1b where the glycosyltransfer reaction is catalyzed by the concerted effect of an active site base to deprotonate the glycosyl acceptor and a divalent metal to assist departure of the leaving group of the glycosyl donor.
D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000032:analysis / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D003619:Dansyl Compounds / Q000737:chemistry; D004789:Enzyme Activation; D029968:Escherichia coli Proteins / Q000032:analysis / Q000737:chemistry*; D005470:Fluorometry / Q000379:methods; D016695:Glycosyltransferases / Q000032:analysis / Q000737:chemistry*; D006602:Hexosyltransferases; D007700:Kinetics; D009097:Multienzyme Complexes / Q000032:analysis / Q000737:chemistry; D002267:Muramoylpentapeptide Carboxypeptidase / Q000032:analysis / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D046915:Penicillin-Binding Proteins; D046928:Peptidoglycan Glycosyltransferase; D010458:Peptidyl Transferases / Q000032:analysis / Q000737:chemistry*; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase; D013379:Substrate Specificity
D001426:Bacterial Proteins; D002352:Carrier Proteins; D003619:Dansyl Compounds; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D046915:Penicillin-Binding Proteins; D011994:Recombinant Proteins; D010458:Peptidyl Transferases; D016695:Glycosyltransferases; D006602:Hexosyltransferases; D046928:Peptidoglycan Glycosyltransferase; C478681:penicillin-binding protein 1B, E coli; C021815:murein transglycosylase; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase; D002267:Muramoylpentapeptide Carboxypeptidase