Site-directed mutagenesis experiments on the putative deprotonation site of squalene-hopene cyclase from Alicyclobacillus acidocaldarius. Site-directed Mutagenesis Experiments on the Putative Deprotonation Site of Squalene-hopene Cyclase from Alicyclobacillus acidocaldarius

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Author(s)

    • Sato Tsutomu SATO Tsutomu
    • Department of Applied Biological Chemistry, Faculty of Agriculture, and Graduate School of Science and Technology, Niigata University
    • Kouda Masanori KOUDA Masanori
    • Department of Applied Biological Chemistry, Faculty of Agriculture, and Graduate School of Science and Technology, Niigata University
    • Hoshino Tsutomu HOSHINO Tsutomu
    • Department of Applied Biological Chemistry, Faculty of Agriculture, and Graduate School of Science and Technology, Niigata University

Abstract

To provide insight into the catalytic mechanism for the final deprotonation reaction of squalene-hopene cyclase (SHC) from <I>Alicyclobacillus acidocaldarius</I>, mutagenesis experiments were conducted for the following ten residues: Thr41, Glu45, Glu93, Arg127, Trp133, Gln262, Pro263, Tyr267, Phe434 and Phe437. An X-ray analysis of SHC has revealed that two types of water molecules (“front water” and “back waters”) were involved around the deprotonation site. The results of these mutagenesis experiments allow us to propose the functions of these residues. The two residues of Gln262 and Pro263 probably work to keep away the isopropyl group of the hopanyl cation intermediate from the “front water molecule,” that is, to place the “front water” in a favorable position, leading to the minimal production of by-products, <I>i.e.</I>, hopanol and hop-21(22)-ene. The five residues of Thr41, Glu45, Glu93, Arg127 and Trp133, by which the hydrogen-bonded network incorporating the “back waters” is constructed, increase the polarization of the “front water” to facilitate proton elimination from the isopropyl moiety of the hopanyl cation, leading to the normal product, hop-22(29)-ene. The three aromatic residues of Tyr267, Phe434 and Phe437 are likely to play an important role in guiding squalene from the enzyme surface to the reaction cavity (substrate channeling) by the strong affinity of their aromatic residues to the squalene substrate.

Journal

  • Bioscience, Biotechnology, and Biochemistry

    Bioscience, Biotechnology, and Biochemistry 68(3), 728-738, 2004-03-23

    Japan Society for Bioscience, Biotechnology, and Agrochemistry

References:  26

Codes

  • NII Article ID (NAID)
    10013143371
  • NII NACSIS-CAT ID (NCID)
    AA10824164
  • Text Lang
    ENG
  • Article Type
    ART
  • ISSN
    09168451
  • NDL Article ID
    6904569
  • NDL Source Classification
    ZR7(科学技術--農林水産--農産) // ZR2(科学技術--生物学--生化学) // ZP1(科学技術--化学・化学工業)
  • NDL Call No.
    Z53-G223
  • Data Source
    CJP  NDL  IR  J-STAGE 
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