Role of arginine residues in the active site of the membrane-bound lytic transglycosylase B from Pseudomonas aeruginosa.
Lytic transglycosylases cleave the beta-(1-->4)-glycosidic bond in the bacterial cell wall heteropolymer peptidoglycan between the N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) residues with the concomitant formation of a 1,6-anhydromuramoyl residue. On the basis of both sequence alignments with and structural considerations of soluble lytic transglycosylase Slt35 from Escherichia coli, four residues were predicted to be involved in substrate binding at the -1 subsite in the soluble derivative of Pseudomonas aeruginosa membrane-bound lytic transglycosylase MltB. These residues were targeted for site-specific replacement, and the effect on substrate binding and catalysis was determined. The residues Arg187 and Arg188, believed to be involved in binding the stem peptide on MurNAc, were shown to play an important role in substrate binding, as evidenced by peptidoglycan affinity assays and SUPREX analysis using MurNAc-dipeptide as ligand. The Michaelis-Menten parameters were determined for the respective mutants using insoluble peptidoglycan as substrate. In addition to affecting the steady-state binding of ligand to enzyme, as indicated by increases in K(M) values, significant decreases in k(cat) values suggested that replacement of either Arg187 and Arg188 with alanine perturbed the stabilization of both the transition state(s) and reaction intermediate. Thus, it appears that Arg187 and Arg188 are vital for proper orientation of the substrate in the active site, and furthermore this supports the proposed role of the stem peptide at binding subsite -2 in catalysis. Replacement of Gln100, a residue that would appear to interact with the N-acetyl group on MurNAc, did not show any changes in substrate affinity or activity.
Reid
Christopher W
CW
Guelph Waterloo Center for Graduate Work in Chemistry and Biochemistry, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Blackburn
Neil T
NT
Clarke
Anthony J
AJ
D000595:Amino Acid Sequence; D001120:Arginine / Q000502:physiology*; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010457:Peptidoglycan / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D016415:Sequence Alignment
D010457:Peptidoglycan; D001120:Arginine; D016695:Glycosyltransferases; C021815:murein transglycosylase