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Identification and characterization of amino acid residues essential for the active site of UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from Staphylococcus aureus.
J Biol Chem. 2006 Jan 20; 281(3):1714-24.JB

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+.

Authors+Show Affiliations

Laboratory of Developmental Biochemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Japan.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

16236703

Citation

Nishida, Satoshi, et al. "Identification and Characterization of Amino Acid Residues Essential for the Active Site of UDP-N-acetylenolpyruvylglucosamine Reductase (MurB) From Staphylococcus Aureus." The Journal of Biological Chemistry, vol. 281, no. 3, 2006, pp. 1714-24.
Nishida S, Kurokawa K, Matsuo M, et al. Identification and characterization of amino acid residues essential for the active site of UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from Staphylococcus aureus. J Biol Chem. 2006;281(3):1714-24.
Nishida, S., Kurokawa, K., Matsuo, M., Sakamoto, K., Ueno, K., Kita, K., & Sekimizu, K. (2006). Identification and characterization of amino acid residues essential for the active site of UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from Staphylococcus aureus. The Journal of Biological Chemistry, 281(3), 1714-24.
Nishida S, et al. Identification and Characterization of Amino Acid Residues Essential for the Active Site of UDP-N-acetylenolpyruvylglucosamine Reductase (MurB) From Staphylococcus Aureus. J Biol Chem. 2006 Jan 20;281(3):1714-24. PubMed PMID: 16236703.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Identification and characterization of amino acid residues essential for the active site of UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from Staphylococcus aureus. AU - Nishida,Satoshi, AU - Kurokawa,Kenji, AU - Matsuo,Miki, AU - Sakamoto,Kimitoshi, AU - Ueno,Kohji, AU - Kita,Kiyoshi, AU - Sekimizu,Kazuhisa, Y1 - 2005/10/19/ PY - 2005/10/21/pubmed PY - 2006/3/18/medline PY - 2005/10/21/entrez SP - 1714 EP - 24 JF - The Journal of biological chemistry JO - J Biol Chem VL - 281 IS - 3 N2 - 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+. SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/16236703/Identification_and_characterization_of_amino_acid_residues_essential_for_the_active_site_of_UDP_N_acetylenolpyruvylglucosamine_reductase__MurB__from_Staphylococcus_aureus_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0021-9258(20)66278-0 DB - PRIME DP - Unbound Medicine ER -