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

该项目的总体目标是确定羟化酶（H6H）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。最终目的是形成一种新的酶，这种酶可以从易接近的tropane衍生物中产生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氧化物。在初步工作中，阿尔伯塔大学的Vederas实验室已经从颠茄（致命的茄属植物）中克隆了H6H，优化了在大肠杆菌中的表达，纯化并表征了这种酶。他们发表的研究表明，这种特殊的酶能迅速地将(-)-阿托品（l -山莨菪碱）氧化为6ß-羟基山莨菪碱，但第二步，即与环氧化物（东莨菪碱）的封闭，保留了新的羟基氧，是缓慢的。另外从产东莨菪碱的植物如Anisodus acutangulus和Hyoscyamus niger （henbane）中克隆并表达了3个序列不同但同源的相应酶。Vederas小组与牛津大学化学系的Christopher J. Schofield教授建立了合作关系，以协助对这些酶进行晶体学研究。

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.