Roles of K151 and D180 in L‐2‐haloacid dehalogenase from Pseudomonas sp. YL: Analysis by molecular dynamics and ab initio fragment molecular orbital calculations |
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Authors: | Takashi Nakamura Azusa Yamaguchi Hirotaka Kondo Hirofumi Watanabe Tatsuo Kurihara Nobuyoshi Esaki Shuichi Hirono Shigenori Tanaka |
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Affiliation: | 1. Laboratory of Molecular Biochemistry, Nagahama Institute of Bio‐Science and Technology, 1266 Tamura‐cho, Nagahama, Shiga 526‐0829, Japan;2. Graduate School of Human Development and Environment, Kobe University, 3‐11 Tsurukabuto, Nada, Kobe 657‐8501, Japan;3. Institute for Chemical Research, Kyoto University, Uji, Kyoto 611‐0011, Japan;4. Laboratory of Physical Chemistry for Drug Design, School of Pharmaceutical Sciences, Kitasato University, 5‐9‐1 Shirokane, Minato‐ku, Tokyo 108‐8641, Japan |
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Abstract: | L ‐2‐haloacid dehalogenase (L ‐DEX) catalyzes the hydrolytic dehalogenation of L ‐2‐haloalkanoic acids to produce the corresponding D ‐2‐hydroxyalkanoic acids. This enzyme is expected to be applicable to the bioremediation of environments contaminated with halogenated organic compounds. We analyzed the reaction mechanism of L ‐DEX from Pseudomonas sp. YL (L ‐DEX YL) by using molecular modeling. The complexes of wild‐type L ‐DEX YL and its K151A and D180A mutants with its typical substrate, L ‐2‐chloropropionate, were constructed by docking simulation. Subsequently, molecular dynamics (MD) and ab initio fragment molecular orbital (FMO) calculations of the complexes were performed. The ab initio FMO method was applied at the MP2/6‐31G level to estimate interfragment interaction energies. K151 and D180, which are experimentally shown to be important for enzyme activity, interact particularly strongly with L ‐2‐chloropropionate, catalytic water, nucleophile (D10), and with each other. Our calculations suggest that K151 stabilizes substrate orientation and balances the charge around the active site, while D180 stabilizes the rotation of the nucleophile D10, fixes catalytic water around D10, and prevents K151 from approaching D10. Further, D180 may activate catalytic water on its own or with K151, S175, and N177. These roles are consistent with the previous results. Thus, MD and ab initio FMO calculations are powerful tools for the elucidation of the mechanism of enzymatic reaction at the molecular level and can be applied to other catalytically important residues. The results obtained here will play an important role in elucidating the reaction mechanism and rational design of L ‐DEX YL with improved enzymatic activity or substrate specificity. © 2009 Wiley Periodicals, Inc. J Comput Chem, 2009 |
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Keywords: | L‐2‐haloacid dehalogenase molecular dynamics calculation ab initio fragment molecular orbital calculation interfragment interaction energy analysis of enzymatic reaction |
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