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J. Biol. Chem., Vol. 281, Issue 3, 1714-1724, January 20, 2006

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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^*

Satoshi Nishida1, Kenji Kurokawa2, Miki Matsuo, Kimitoshi Sakamoto, Kohji Ueno¶, Kiyoshi Kita, and Kazuhisa Sekimizu

From the Laboratory of Developmental Biochemistry, Graduate School of Pharmaceutical Sciences, the Department of Biomedical Chemistry, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033, Japan and the ^¶Laboratory of Microbiology, Research Institute of Pharmaceutical Sciences, Musashino University, Tokyo 202-8585, Japan

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⁺.

Received for publication, August 23, 2005 , and in revised form, September 28, 2005.

^* This work was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science and by the Industrial Technology Research Grant Program in '04 from the New Energy and Industrial Technology Development Organization of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Laboratory of Microbiology, Research Institute of Pharmaceutical Sciences, Musashino University, Tokyo 202-8585, Japan.

² To whom correspondence should be addressed: Laboratory of Developmental Biochemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel.: 81-3-5841-4821; Fax: 81-3-5684-2973; E-mail: kurokawa{at}mol.f.u-tokyo.ac.jp.

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