Lichman Benjamin R, Gershater Markus C, Lamming Eleanor D, Pesnot Thomas, Sula Altin, Keep Nicholas H, Hailes Helen C, Ward John M
Department of Biochemical Engineering, University College London, UK.
FEBS J. 2015 Mar;282(6):1137-51. doi: 10.1111/febs.13208. Epub 2015 Feb 9.
Norcoclaurine synthase (NCS) (EC 4.2.1.78) catalyzes the Pictet-Spengler condensation of dopamine and an aldehyde, forming a substituted (S)-tetrahydroisoquinoline, a pharmaceutically important moiety. This unique activity has led to NCS being used for both in vitro biocatalysis and in vivo recombinant metabolism. Future engineering of NCS activity to enable the synthesis of diverse tetrahydroisoquinolines is dependent on an understanding of the NCS mechanism and kinetics. We assess two proposed mechanisms for NCS activity: (a) one based on the holo X-ray crystal structure and (b) the 'dopamine-first' mechanism based on computational docking. Thalictrum flavum NCS variant activities support the dopamine-first mechanism. Suppression of the non-enzymatic background reaction reveals novel kinetic parameters for NCS, showing it to act with low catalytic efficiency. This kinetic behaviour can account for the ineffectiveness of recombinant NCS in in vivo systems, and also suggests NCS may have an in planta role as a metabolic gatekeeper. The amino acid substitution L76A, situated in the proposed aldehyde binding site, results in the alteration of the enzyme's aldehyde activity profile. This both verifies the dopamine-first mechanism and demonstrates the potential for the rational engineering of NCS activity.
去甲乌药碱合酶（NCS）（EC 4.2.1.78）催化多巴胺与醛的 Pictet-Spengler 缩合反应，形成一种取代的（S）-四氢异喹啉，这是一种具有重要药学意义的部分。这种独特的活性使得 NCS 被用于体外生物催化和体内重组代谢。未来对 NCS 活性进行工程改造以实现多种四氢异喹啉的合成，取决于对 NCS 机制和动力学的理解。我们评估了两种关于 NCS 活性的 proposed 机制：（a）一种基于全酶 X 射线晶体结构，（b）基于计算对接的“多巴胺优先”机制。唐松草 NCS 变体活性支持多巴胺优先机制。非酶背景反应的抑制揭示了 NCS 的新动力学参数，表明其催化效率较低。这种动力学行为可以解释重组 NCS 在体内系统中的无效性，也表明 NCS 可能在植物中作为代谢守门人发挥作用。位于 proposed 醛结合位点的氨基酸取代 L76A 导致酶的醛活性谱发生改变。这既验证了多巴胺优先机制，也证明了对 NCS 活性进行合理工程改造的潜力。 （注：原文中“proposed”未准确翻译，结合语境应为“提出的”之类意思）
