Neisseria gonorrheae O-acetylpeptidoglycan esterase, a serine esterase with a Ser-His-Asp catalytic triad.
O-Acetylpeptidoglycan esterase from Neisseria gonorrheae FA1090 is similar in sequence to family CE-3 carbohydrate esterases of the CAZy classification system, and it functions to release O-linked acetyl groups from the C-6 position of muramoyl residues in O-acetylated peptidoglycan. Here, we characterize the peptidoglycan of N. gonorrheae FA1090 as being O-acetylated and find that it serves as a substrate for the esterase. The influence of pH on the activity of O-acetylpeptidoglycan esterase was determined, and pKa values of 6.38 and 6.78 for the enzyme-substrate complex (VEt-1) and free enzyme (VEt-1KM-1), respectively, were calculated. The enzyme was inactivated by sulfonyl fluorides but not by EDTA. Multiple-sequence alignment of the O-acetylpeptidoglycan esterase family 1 enzymes with members of the CE-3 enzymes and protein modeling studies identified Ser80, Asp366, and His369 as three invariant amino acid residues that could potentially serve as a catalytic triad. Replacement of each with alanine was accomplished by site-directed mutagenesis, and the resulting mutant proteins were purified to apparent homogeneity. The specific activity of each of the three esterase derivatives was greatly reduced on O-acetylpeptidoglycan. Using the artificial substrate p-nitrophenyl acetate, a kinetic analysis revealed that the turnover number (VEt-1) but not KM was affected by the replacements. These data thus indicate that N. gonorrheae O-acetylpeptidoglycan esterase, and by analogy the CE-3 family of enzymes, function as serine esterases involving a Ser-His-Asp catalytic triad.
Weadge
Joel T
JT
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Clarke
Anthony J
AJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D004950:Esterases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009344:Neisseria gonorrhoeae / Q000201:enzymology*; D010457:Peptidoglycan / Q000737:chemistry; D016415:Sequence Alignment
D010457:Peptidoglycan; D004950:Esterases; C520356:O-acetylpeptidoglycan esterase, Neisseria gonorrheae