'Dopamine-first' mechanism enables the rational engineering of the norcoclaurine synthase aldehyde activity profile.
'Dopamine-first' mechanism enables the rational engineering of the norcoclaurine synthase aldehyde activity profile.
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DOI：
10.1111/febs.13208
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发表时间：
2015-03
期刊：
影响因子：
--
通讯作者：
Ward JM
中科院分区：
文献类型：
--
作者：
Lichman BR;Gershater MC;Lamming ED;Pesnot T;Sula A;Keep NH;Hailes HC;Ward JM
去甲乌药碱合酶（NCS）（EC 4.2.1.78）催化多巴胺和醛的Pictet-Spengler缩合，形成取代的（S）-四氢异喹啉，一种药学上重要的部分。这种独特的活性导致NCS被用于体外生物催化和体内重组代谢。NCS活性的未来工程化以使得能够合成不同的四氢异喹啉取决于对NCS机制和动力学的理解。我们评估了NCS活性的两种拟议机制：（a）一种基于全息X射线晶体结构和（B）基于计算对接的“多巴胺优先”机制。黄曲草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，表明它以低催化效率起作用。这种动力学行为可以解释重组NCS在体内系统中的无效性，并且还表明NCS可能具有作为代谢看门人的体内作用。氨基酸取代L76 A，位于建议的醛结合位点，导致酶的醛活性曲线的改变。这既验证了多巴胺优先机制，又证明了NCS活性的合理工程潜力。
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.
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