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Hyoscyamine-6ü-羟基酶(H6H)的结构和机制，并进行位点特异性突变以扩大其底物专一性。最终的目标是形成新的酶，既可以从可获得的托烷衍生物中产生改变的东莨菪碱类似物，也可以将各种环烷烃氧化成相应的有用的环氧化物。在初步工作中，艾伯塔大学的Vederas实验室已经从颠茄(致命的茄子)中克隆了H6H，并对该酶进行了纯化和鉴定。他们发表的工作表明，这种特殊的酶能迅速将(-)-阿托品(L-山梨碱)氧化成6-羟基山梨胺，但第二步，即关闭环氧化物(东莨菪碱)并保留新的羟基氧，速度较慢。此外，还克隆和表达了另外三种具有不同但同源序列的相应酶，这些酶来自产生东莨菪碱的植物，如尖角异尖异尖线虫和黑花蛇舌草。Vederas小组已经与牛津大学化学系的Christopher J.Schofield教授建立了合作关系，以协助对这些酶的结晶学研究。

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.