Identification and Characterization of Amino Acid Residues Essential for the Active Site of Peptidoglycan Biosynthesis Enzymes from Staphylococcus aureus

Abstract

The enzymes essential for bacterial peptidoglycan biosynthesis are attractive targets for antimicrobial drug development. One of these is MurB, which contains FAD as a cofactor and catalyzes the NADPH-dependent reduction of UDP-N-acetylenolpyruvylglucosamine (UDP-GlcNAcEP) to UDP-N-acetylmuramic acid. This study examined the roles of the conserved amino acid residues of Staphylococcus aureus MurB, which are located near the active site in X-ray crystal structures. Seven out of 11 site-directed mutated murB genes lost the ability to complement a temperature-sensitive S. aureus murB mutant. Biochemical characterization of the seven mutated MurB proteins revealed that they cannot carry out the reduction of UDP-GlcNAcEP, though they can all catalyze the intramolecular reduction of FAD via NADPH. Spectrometric analyses of the oxidized form of the mutated proteins in the presence and absence of NADP+ or UDP-GlcNAcEP revealed that these essential amino acid residues play four distinct roles in substrate interactions. An essential residue of MurC, which catalyzes a ligation of L-Ala to UDP-N-acetylmuramic acid was also revealed by a chemical mutagenesis approach.