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Journal of Biochemistry Advance Access originally published online on December 15, 2007
Journal of Biochemistry 2008 143(3):417-424; doi:10.1093/jb/mvm237
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© 2007 The Japanese Biochemical Society.

Staphylococcus aureus MurC Participates in L-Alanine Recognition via Histidine 343, a Conserved Motif in the Shallow Hydrophobic Pocket

Kenji Kurokawa1,*,{dagger}, Satoshi Nishida1,2,{dagger}, Mihoko Ishibashi1, Hikaru Mizumura1, Kohji Ueno2, Takashi Yutsudo3, Hideki Maki3, Kazuhisa Murakami3 and Kazuhisa Sekimizu1

1Laboratory of Microbiology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033; 2Laboratory of Microbiology, Research Institute of Pharmaceutical Sciences, Musashino University, Tokyo 202-8585; and 3Discovery Research Laboratories, Shionogi & Co., Ltd., Osaka 561-0825, Japan

*To whom correspondence should be addressed. Tel: +81 3 5841 4821, Fax: +81 3 5684 2973, E-mail: kurokawa{at}mol.f.u-tokyo.ac.jp

Received October 26, 2007; Accepted November 26, 2007


  Abstract

UDP-N-acetylmuramic acid:L-alanine ligase that is encoded by the murC gene, is indispensable for bacterial peptidoglycan biosynthesis and an important target for the development of antibacterial agents. Structure of MurC ligase with substrates has been described, however, little validation via studying the effects of mutations on the structure of MurC has been performed. In this study, we carried out a functional in vitro and in vivo characterization of Staphylococcus aureus MurCH343Y protein that has a temperature-sensitive mutation of a conserved residue in the predicted shallow hydrophobic pocket that holds a short L-alanine side chain. Purified H343Y and wild-type MurC had Km values for L-alanine of 3.2 and 0.44 mM, respectively, whereas there was no significant difference in their Km values for ATP and UDP-N-acetylmuramic acid, suggesting the specific alteration of L-alanine recognition in MurCH343Y protein. In a synthetic medium that excluded L-alanine, S. aureus murCH343Y mutant cells showed an allele-specific slow growth phenotype that was suppressed by addition of L-alanine. These results suggest that His343 of S. aureus MurC is essential for high-affinity binding to L-alanine both in vitro and in vivo and provide experimental evidence supporting the structural information of MurC ligase.

Key Words: Gram-positive bacteria, MurC, peptidoglycan, Staphylococcus aureus, UDP-N-acetylmuramic acid:L-alanine ligase

Abbreviations: UDP-MurNAc, UDP-N-acetylmuramic acid

{dagger}These two authors contributed equally to this work.


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