Identification and characterization of amino acid residues essential for the active site of UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from Staphylococcus aureus.
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+.
Nishida
Satoshi
S
Laboratory of Developmental Biochemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Japan.
Kurokawa
Kenji
K
Matsuo
Miki
M
Sakamoto
Kimitoshi
K
Ueno
Kohji
K
Kita
Kiyoshi
K
Sekimizu
Kazuhisa
K
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry* / Q000378:metabolism; D017124:Conserved Sequence; D017931:DNA Primers / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013211:Staphylococcus aureus / Q000201:enzymology*
D001426:Bacterial Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; D002237:Carbohydrate Dehydrogenases; C023215:UDP-N-acetylmuramate dehydrogenase