State Key Laboratory of Bio-organic and Natural Products Chemistry, Center for Excellence in Molecular Synthesis, Shanghai Institute of Organic Chemistry, University of Chinese Academy of Sciences, Shanghai, China.
Department of Chemistry and Biochemistry, Department of Chemical and Biomolecular Engineering, University of California Los Angeles, Los Angeles, CA, USA.
Nat Chem. 2019 Sep;11(9):812-820. doi: 10.1038/s41557-019-0294-x. Epub 2019 Jul 22.
LepI is an S-adenosylmethionine (SAM)-dependent pericyclase that catalyses the formation of the 2-pyridone natural product leporin C. Biochemical characterization has shown that LepI can catalyse stereoselective dehydration to yield a reactive (E)-quinone methide that can undergo bifurcating intramolecular Diels-Alder (IMDA) and hetero-Diels-Alder (HDA) cyclizations from an ambimodal transition state, as well as a [3,3]-retro-Claisen rearrangement to recycle the IMDA product into leporin C. Here, we solve the X-ray crystal structures of SAM-bound LepI and in complex with a substrate analogue, the product leporin C, and a retro-Claisen reaction transition-state analogue to understand the structural basis for the multitude of reactions. Structural and mutational analysis reveals how nature evolves a classic methyltransferase active site into one that can serve as a dehydratase and a multifunctional pericyclase. Catalysis of both sets of reactions employs H133 and R295, two active-site residues that are not found in canonical methyltransferases. An alternative role of SAM, which is not found to be in direct contact with the substrate, is also proposed.
LepI 是一种依赖 S-腺苷甲硫氨酸 (SAM) 的周质环化酶，可催化 2-吡啶酮天然产物 Leporin C 的形成。生化特性研究表明，LepI 可以催化立体选择性脱水，生成活性 (E)-醌甲亚胺，该中间体能从双模态过渡态经历分叉的分子内 Diels-Alder (IMDA) 和杂 Diels-Alder (HDA) 环化，以及 [3,3]-逆 Claisen 重排，将 IMDA 产物循环回 Leporin C。在此，我们解析了 SAM 结合的 LepI 及其与底物类似物、产物 Leporin C 以及逆 Claisen 反应过渡态类似物的 X 射线晶体结构，以了解多种反应的结构基础。结构和突变分析揭示了自然界如何将经典的甲基转移酶活性位点进化为既能作为脱水酶又能作为多功能周质环化酶的活性位点。两套反应的催化都利用了 H133 和 R295，这两个活性位点残基在典型的甲基转移酶中不存在。还提出了 SAM 的替代作用，它与底物没有直接接触。
