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
复制标题
DOI：
10.1271/bbb.68.728
复制
发表时间：
2004-03-01
影响因子：
1.6
通讯作者：
Hoshino, T
中科院分区：
文献类型：
--
作者：
Sato, T;Kouda, M;Hoshino, T
为了深入了解酸热脂环酸芽孢杆菌角鲨烯-藿烯环化酶（SHC）最终去质子化反应的催化机制，对以下十个残基进行了诱变实验：Thr41、Glu45、Glu93、Arg127、Trp133、Gln262、Pro263、Tyr267、Phe434和Phe437。 SHC 的 X 射线分析表明，去质子化位点周围存在两种类型的水分子（“前水”和“后水”）。这些诱变实验的结果使我们能够提出这些残基的功能。 Gln262和Pro263这两个残基可能起到使藿基阳离子中间体的异丙基远离“前水分子”的作用，即将“前水”置于有利的位置，从而使副产物（即藿醇和hop-21(22)-ene）的产生最少。 Thr41、Glu45、Glu93、Arg127 和 Trp133 五个残基构建了包含“后水”的氢键网络，增加了“前水”的极化，以促进质子从藿基阳离子的异丙基部分消除，产生正常产物 hop22(29)-ene。 Tyr267、Phe434和Phe437这三个芳香族残基很可能通过其芳香族残基与角鲨烯底物的强亲和力，在引导角鲨烯从酶表面进入反应腔（底物通道）中发挥重要作用。
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, hop22(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.