Site-directed Mutagenesis Experiments on the Putative Deprotonation Site of Squalene-hopene Cyclase from<i>Alicyclobacillus acidocaldarius</i>

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

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  • Site-directed Mutagenesis Experiments on the Putative Deprotonation Site of Squalene-hopene Cyclase from Alicyclobacillus acidocaldarius

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To provide insight into the catalytic mechanism for the final deprotonation reaction of squalene-hopene cyclase (SHC) from Alicyclobacillus acidocaldarius, 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.e., 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.

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