O-acetylation of peptidoglycan: Genomic identification and biochemical characterization of the enzymes involved
Peptidoglycan, an essential component of most eubacterial cell walls, provides osmotic stability and an exo-skeleton through which cellular shape is imparted. Addition of acetate to the C6 position of ' N'-acetylmuramic acid of peptidoglycan has been demonstrated to affect the activity of muramidases, like lysozyme, as well as endogenous lytic transglycosylases. This later group of enzymes appear to be ubiquitous in peptidoglycan synthesizing bacteria and are responsible for the growth and development of the peptidoglycan sacculus. Without the removal of O-linked acetate, these processes would be severely limited. Despite this central importance, there is little information regarding peptidoglycan O-acetylation and even less known about its de-acetylation. The current research is the first to identify a potential cluster of O-acetylpeptidoglyca nrelated genes. They are encoded in the genomes of a variety of Gram-negative and Gram-positive bacteria, including a number of important human pathogens, such as species of 'Neisseria, Helicobacter, Campylobacter', and 'Bacillus'. One of the genes from this cluster, 'pat', is similar to the membrane-bound O-acetyltransferase family of proteins and is proposed to function as a peptidoglycan acetyltransferase. The other two genes in the cluster, 'ape1a' and 'ape2 ', have been identified as acetylpeptidoglycan esterases (Ape). Herein, studies were performed to characterize the function of these proteins. Data from activity assays involving HPLC-based organic acid analysis, NMR, and chromogenic detection methods confirmed that Ape1a from 'Neisseria gonorrhoeae' is the first peptidoglycan de-O-acetylase to be identified and characterized. Through a combination of sequence alignments, three-dimensional modeling, site-directed mutagenesis, Michaelis-Menten kinetics and inhibition studies, a number of residues in Ape1a (Gly78, Ser80, Asn235, Asn268, Thr267, Asp 366 and His369) have been implicated in the role of substrate binding and catalysis. Of particular importance is the identification of the Ser80/His369/Asp366 catalytic triad of Ape1a, which is characteristic of the serine protease/esterase/lipase family of proteins. These studies have aided in understanding the mechanism by which bacteria maintain certain levels of O-acetylated peptidoglycan and how these levels affect cellular processes, like cell-wall restructuring and turnover. Furthermore, these results are significant given the increasing number of pathogenic bacteria that are known to perform this O-acetyl modification.