Aspartic acid residues in BBE- like enzymes from Morus alba promote a function shift from oxidative cyclization to dehydrogenation

• 成果类型: Article
• 署名作者: Guo, Nianxin; Gu, Jun; Zhou, Qingyang; Liu, Fang; Dong, Haoran; Ding, Qi; Wang, Qixuan; Wu, Dongshan; Yang, Jun; Fan, Junping; Gao, Lei; Houk, Kendall N.; Lei, Xiaoguang
• 署名单位: Peking University; Peking University; Chinese Academy of Sciences; Peking University; Nanjing University of Science & Technology; University of California System; University of California Los Angeles; Nanjing Agricultural University; Wuhan University
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-14741
• DOI: 10.1073/pnas.2504346122
• 发表日期: 2025-08-26
• 关键词: berberine bridge enzyme; molecular-dynamics simulations; gaussian-type basis; orbital methods; atomic charges; amber; specificity; mechanism; synthase; FAMILY

摘要

Berberine bridge enzyme (BBE)-like enzymes catalyze various oxidative cyclization and dehydrogenation reactions in natural product biosynthesis, but the molecular mechanism underlying the selectivity remains unknown. Here, we elucidated the catalytic mechanism of BBE-like oxidases from Morus alba involved in the oxidative cyclization and dehydrogenation of moracin C. X-ray crystal structures of a functionally promiscuous flavin adenine dinucleotide (FAD)-bound oxidase, MaDS1, with and without an oxidative dehydrogenation product were determined at 2.03 & Aring; and 2.21 & Aring; resolution, respectively. Structure-guided mutagenesis and sequence analysis have identified a conserved aspartic acid that directs the reaction toward the oxidative dehydrogenation pathway. A combination of density functional theory (DFT) calculations and molecular dynamics (MD) simulations has revealed that aspartic acid acts as the catalytic base to deprotonate the carbon-cation intermediate to generate the dehydrogenated product, which otherwise undergoes a spontaneous 6 pi electrocyclization in the oxidative cyclization pathway to furnish the 2H-benzopyran product.