Lichman Benjamin R, Sula Altin, Pesnot Thomas, Hailes Helen C, Ward John M, Keep Nicholas H
Department of Biochemical Engineering, University College London , Gower Street, London WC1E 6BT, U.K.
Institute for Structural and Molecular Biology, Department of Biological Sciences, Birkbeck, University of London , Malet Street, London WC1E 7HX, U.K.
Biochemistry. 2017 Oct 10;56(40):5274-5277. doi: 10.1021/acs.biochem.7b00769. Epub 2017 Sep 20.
Norcoclaurine synthase (NCS) is a Pictet-Spenglerase that catalyzes the first key step in plant benzylisoquinoline alkaloid metabolism, a compound family that includes bioactive natural products such as morphine. The enzyme has also shown great potential as a biocatalyst for the formation of chiral isoquinolines. Here we present new high-resolution X-ray crystallography data describing Thalictrum flavum NCS bound to a mechanism-inspired ligand. The structure supports two key features of the NCS "dopamine-first" mechanism: the binding of dopamine catechol to Lys-122 and the position of the carbonyl substrate binding site at the active site entrance. The catalytically vital residue Glu-110 occupies a previously unobserved ligand-bound conformation that may be catalytically significant. The potential roles of inhibitory binding and alternative amino acid conformations in the mechanism have also been revealed. This work significantly advances our understanding of the NCS mechanism and will aid future efforts to engineer the substrate scope and catalytic properties of this useful biocatalyst.
去甲乌药碱合酶（NCS）是一种 Pictet-Spengler 酶，催化植物苄基异喹啉生物碱代谢的第一步关键反应，该化合物家族包括吗啡等生物活性天然产物。该酶作为手性异喹啉形成的生物催化剂也显示出巨大潜力。在此，我们展示了新的高分辨率 X 射线晶体学数据，描述了与一种受机制启发的配体结合的黄唐松草 NCS。该结构支持 NCS“多巴胺优先”机制的两个关键特征：多巴胺儿茶酚与 Lys-122 的结合以及羰基底物结合位点在活性位点入口处的位置。催化至关重要的残基 Glu-110 占据了一个以前未观察到的与配体结合的构象，这可能具有催化意义。还揭示了抑制性结合和替代氨基酸构象在该机制中的潜在作用。这项工作极大地推进了我们对 NCS 机制的理解，并将有助于未来设计这种有用生物催化剂的底物范围和催化特性的努力。
