Structure and Mechanism of Hyoscyamine-6ß-Hydroxylase (H6H), an Enzyme that converts (-)-Atropine to Scopolamine: Prospects for Aerobic Epoxidation of Non-Activated Alkane Chains

本项目的总体目标是确定莨菪碱-6 β-羟化酶（H6 H）的结构和机制，并进行位点特异性突变以扩大其底物特异性。最终目标是形成新的酶，其可以从可获得的托烷衍生物制备改变的东莨菪碱类似物，或者将各种环烷烃氧化成相应的有用的异丁酰亚胺。在初步工作中，阿尔伯塔大学的Vederas实验室已经从颠茄中克隆了H6 H，并优化了其在E.大肠杆菌，纯化和表征的酶。他们已发表的研究表明，这种特殊的酶能迅速将（-）-阿托品（L-莨菪碱）氧化为6 β-羟基莨菪碱，但第二步，即封闭为环氧化物（东莨菪碱）并保留新的羟基氧，是缓慢的。另外三个相应的酶与不同的，但同源序列的东莨菪碱生产的植物，如三分之一acutangulus和尼日尔（天仙子）也被克隆和表达。Vederas小组与牛津大学化学系的Christopher J.斯科菲尔德教授建立了合作关系，以协助这些酶的晶体学研究。

The overall goal of this project is to determine the structure and mechanism of hyoscyamine-6ß-hydroxylase (H6H) and to do site specific mutations to expand its substrate specificity. The ultimate objective is to form new enzymes that can either make altered scopolamine analogs from accessible tropane derivatives or oxidize a variety of cyclic alkanes to corresponding useful expoxides. In preliminary work, the Vederas laboratory at the University of Alberta has already cloned H6H from Atropa belladonna (deadly nightshade) optimized for expression in E. coli, purified and characterized the enzyme. Their published work shows that this particular enzyme oxidized (-)-atropine (L-hyoscyamine) to 6ß-hydroxyhyoscyamine rapidly, but that the second step, namely closure to the epoxide (scopolamine) with retention of the new hydroxyl oxygen, is slow. Three additional corresponding enzymes with different but homologous sequences from scopolamine-producing plants such as Anisodus acutangulus and Hyoscyamus niger (henbane) have also been cloned and expressed. A collaboration has been established by the Vederas group with Prof. Christopher J. Schofield of the Chemistry Department at Oxford University to assist in crystallographic studies of these enzymes.