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 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, although 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: Arg213 is essential for maintenance of the electronic state of FAD; Arg176 is required for interaction with UDP-GlcNAcEP; His259 is required for interaction with both UDP-GlcNAcEP and NADP+; and Asn71, Tyr175, Ser226, and Glu296 are not apparently required for interaction with either ligand. The results presented here identify for the first time the amino acid residues of MurB that are required for the interaction with UDP-Glc-NAcEP and NADP+.
Highlights
Peptidoglycan consists of a saccharide backbone that is formed by alternating N-acetylglucosamine and N-acetylmuramic acid residues with tetra- or pentapeptides that branch from N-acetylmuramic acid [1,2,3]
We examined whether the amino acid residues of S. aureus MurB, which are well conserved among various bacterial MurB proteins and are located close to the catalytic site, are essential for catalytic activity
Ser226 corresponds to Ser229 of E. coli MurB, which was reported previously as a catalytic residue whose hydroxyl group is thought to transfer a proton for the reduction of UDP-GlcNAcEP [18]
Summary
Roles in the enzyme activity as well as protein structural information is critical for developing enzyme-specific antibacterial drugs. Amino acid residues located in or close to substrate binding sites, which are revealed by x-ray or NMR structural analysis, are not always essential for the enzyme activity. We examined whether the amino acid residues of S. aureus MurB, which are well conserved among various bacterial MurB proteins and are located close to the catalytic site, are essential for catalytic activity. The roles of each essential residue in the interaction of MurB with NADPϩ and UDPGlcNAcEP were examined by differential spectral analyses of intramolecular FAD. This allowed classification of the essential residues into four functional groups
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