Laboratoire de Physiologie Cellulaire et Végétale, Université Grenoble Alpes, CNRS UMR5168, CEA/DRF/BIG, INRA UMR 1417, 17, Avenue des Martyrs, 38054 Grenoble, France.
Plant J. 2016 Sep;87(6):641-53. doi: 10.1111/tpj.13225. Epub 2016 Aug 6.
Growing pharmaceutical interest in benzylisoquinoline alkaloids (BIA) coupled with their chemical complexity make metabolic engineering of microbes to create alternative platforms of production an increasingly attractive proposition. However, precise knowledge of rate-limiting enzymes and negative feedback inhibition by end-products of BIA metabolism is of paramount importance for this emerging field of synthetic biology. In this work we report the structural characterization of (S)-norcoclaurine-6-O-methyltransferase (6OMT), a key rate-limiting step enzyme involved in the synthesis of reticuline, the final intermediate to be shared between the different end-products of BIA metabolism, such as morphine, papaverine, berberine and sanguinarine. Four different crystal structures of the enzyme from Thalictrum flavum (Tf 6OMT) were solved: the apoenzyme, the complex with S-adenosyl-l-homocysteine (SAH), the complexe with SAH and the substrate and the complex with SAH and a feedback inhibitor, sanguinarine. The Tf 6OMT structural study provides a molecular understanding of its substrate specificity, active site structure and reaction mechanism. This study also clarifies the inhibition of Tf 6OMT by previously suggested feedback inhibitors. It reveals its high and time-dependent sensitivity toward sanguinarine.
医药领域对苯并异喹啉生物碱（BIA）的浓厚兴趣，加上其化学结构的复杂性，使得微生物代谢工程成为创造替代生物合成平台的一个极具吸引力的方案。然而，对于这一新兴的合成生物学领域来说，准确了解限速酶以及 BIA 代谢终产物的负反馈抑制作用至关重要。在这项工作中，我们报告了（S）-去甲荷叶碱-6-O-甲基转移酶（6OMT）的结构特征，该酶是参与荷叶碱合成的关键限速酶，荷叶碱是 BIA 代谢的不同终产物（如吗啡、罂粟碱、小檗碱和血根碱）共有的最终中间体。我们从唐松草属（Tf 6OMT）中解析了该酶的四个不同晶体结构：apo 酶、与 S-腺苷-L-同型半胱氨酸（SAH）的复合物、与 SAH 和底物的复合物，以及与 SAH 和反馈抑制剂血根碱的复合物。Tf 6OMT 的结构研究提供了对其底物特异性、活性位点结构和反应机制的分子理解。这项研究还澄清了先前提出的反馈抑制剂对 Tf 6OMT 的抑制作用。它揭示了 Tf 6OMT 对血根碱的高敏感性和时依赖性。
