Luk Louis Y P, Bunn Shannon, Liscombe David K, Facchini Peter J, Tanner Martin E
Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1.
Biochemistry. 2007 Sep 4;46(35):10153-61. doi: 10.1021/bi700752n. Epub 2007 Aug 15.
Norcoclaurine synthase catalyzes an asymmetric Pictet-Spengler condensation of dopamine and 4-hydroxyphenylacetaldehyde to give (S)-norcoclaurine. This is the first committed step in the biosynthesis of the benzylisoquinoline alkaloids that include morphine and codeine. In this work, the gene encoding for the Thalictrum flavum norcoclaurine synthase is highly overexpressed in Escherichia coli and the resulting His-tagged recombinant enzyme is purified for the first time. A continuous assay based on circular dichroism spectroscopy is developed and used to monitor the kinetics of the enzymatic reaction. Dopamine analogues bearing a methoxy or hydrogen substituent in place of the C-1 phenolic group were readily accepted by the enzyme whereas those bearing the same substituents at C-2 were not. This supports a mechanism involving a two-step cyclization of the putative iminium ion intermediate that does not proceed via a spirocyclic intermediate. The reaction of [3,5,6-2H]dopamine was found to be slowed by a kinetic isotope effect of 1.7 +/- 0.1 on the value of kcat/KM. This is interpreted as showing that the deprotonation step causing rearomatization is partially rate determining in the overall reaction.
去甲乌药碱合酶催化多巴胺和4-羟基苯乙醛的不对称Pictet-Spengler缩合反应，生成（S）-去甲乌药碱。这是包括吗啡和可待因在内的苄基异喹啉生物碱生物合成中的第一个关键步骤。在这项工作中，编码唐松草去甲乌药碱合酶的基因在大肠杆菌中高度过表达，首次纯化得到了带有His标签的重组酶。开发了一种基于圆二色光谱的连续测定法，并用于监测酶促反应的动力学。该酶很容易接受在C-1酚羟基位置带有甲氧基或氢取代基的多巴胺类似物，而在C-2位置带有相同取代基的类似物则不被接受。这支持了一种机制，即假定的亚胺离子中间体的两步环化反应不通过螺环中间体进行。发现[3,5,6-2H]多巴胺的反应因动力学同位素效应使kcat/KM值降低了1.7±0.1。这被解释为表明导致重新芳构化的去质子化步骤在整个反应中部分决定反应速率。
