Identification of the namH gene encoding the hydroxylase responsible for the N-glycolylation of the mycobacterial peptidoglycan.

摘要

The peptidoglycan of most bacteria consists of a repeating disaccharide unit of beta-1,4 linked N-acetylmuramic acid and N-acetylglucosamine. However, the muramic acid moieties of the mycobacterial peptidoglycan are N-glycolylated, not N-acetylated. This is a rare modification, seen only in the peptidoglycan of mycobacteria and five other closely related genera of bacteria. The N-glycolylation of sialic acids is a unique carbohydrate modification that has been studied extensively in mammals. However, the significance of the N-glycolylation of bacterial peptidoglycan is unknown.; It has been proposed that a soluble hydroxylase enzyme converts UDP- N-acetylmuramic acid to UDP-N-glycolylmuramic acid during the cytoplasmic stage of peptidoglycan synthesis. In support of this hypothesis, a soluble UDP-N-acetylmuramic acid hydroxylase activity was discovered many years ago in Nocardia asteroides, but no protein was purified and no gene was identified.; The goal of this project was to identify the gene encoding the hydroxylase responsible for the N-glycolylation of the mycobacterial peptidoglycan. We developed a novel assay for the mycobacterial UDP- N-acetylmuramic acid hydroxylation reaction and demonstrated that Mycobacterium smegmatis has an enzyme activity that can convert UDP- N-acetylmuramic acid to UDP-N-glycolylmuramic acid.; We identified the gene encoding the mycobacterial UDP-N-acetylmuramic acid hydroxylase by computer database searching and motif comparisons with the mammalian enzymes responsible for the N-glycolylation of sialic acids. We have named the mycobacterial gene namH. The gene is not essential for in vitro growth as we were successful in deleting the names gene in M. smegmatis. The M. smegmatis mutant is devoid of UDP-N-acetylmuramic acid hydroxylase activity. Furthermore, the mutant exhibits increased susceptibility to beta-lactam antibiotics and lysozyme. Our studies suggest that the N-glycolylation of mycobacterial peptidoglycan may play a role in lysozyme resistance or may contribute to the structural stability of the cell wall architecture.