Bioorganic studies on the recogniton mechanism of the methyl groups of the substrate skeleton by squalene and oxidosqualene cyclases

本研究从底物类似物和酶化学两个方面深入研究了霍烯、四氢吡喃醇和羊毛甾醇脱氢酶催化角鲨烯和氧化角鲨烯环化反应的机理，获得了以下新的结果：1.角鲨烯环化酶在大肠杆菌中的高效表达。Biotechnol. Biochem. 62，pp. 407-411（1998）。以色氨酸残基为目标的定点突变实验表明，被一些工作者认为是活性位点的重复保守序列，即QW基序，不是活性位点，并且具有特异的抗热变性功能。Biosci. Biotechnol. Biochem.63，（1999），in press. W 489 F和W169 H的点突变使我们提出了一种新的环化机制，即在多环化过程中发生了从5元D环到6元D环的扩环。J.Chem. Soc. Chem. Commun. 2617-2618（1998）。底物类似物表明，在羊毛甾醇合酶的情况下，在C-环形成状态下也发生扩环反应。1591-1592（1998）号决议。末端甲基对于角鲨烯底物的正确折叠对于五元B-环的形成和对于提交供公开的多环化反应的引发都是关键的。

This research project has gained insights into the cyclization mechanism of squalene and oxidosqualene catalyzed by hopene, tetrahymanol and lanosterol synthases.Investigations from the two approaches of the substrate analogues and the enzymic chemistry have revealed the following new results :1. Overexpression of squalene-hopene cyclase in Escherichia coli Biosci. Biotechnol. Biochem. 62, pp. 407-411(1998).2. Site-directed mutagenesis experiments, targetted for tryptophan residues, revealed that the repetitive conserved alighnments, i.e. QW motifs, which has been presumed to be active sites by some workers, were not active sites and had a specific function against thermal denaturation. Biosci. Biotechnol. Biochem. 63, (1999), in press.3. Point mutations of W489F and W l 69H has led us to propose the new cyclization mechanism, that is, a ring expansion from 5- to 6-membered D-ring occur during the polycyclization. J.Chem. Soc. Chem. Commun. 2617-2618 (1998).4. The substrate analogues showed that a ring expansion reaction ocurs also at the state of C-ring formation in the case of lanosterol synthase.J.Chem. Soc. Chem. Commun. 1591-1592 (1998).5. the terminal methyl groups are critical to the correct folding of squalene substrate both for the formation of the five-membered B-ring and for the initiation of the polycyclization reaction submitted for publication.