biostudies-literatureGuo NUnited States NSFMOST | National Key Research and Development Program of China (NKPs)CAS | ICCAS | Beijing National Laboratory for Molecular Sciences (BNLMS)National Natural Science Foundation of China| National Natural Science Foundation of Chinae2504346122https://www.ebi.ac.uk/biostudies/studies/S-EPMC12403149biostudies-literatureUnknown122(34)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 <i>Morus alba</i> 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 Å and 2.21 Å 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 functProceedings of the National Academy of Sciences of the United States of AmericaAspartic acid residues in BBE-like enzymes from &lt;i&gt;Morus alba&lt;/i&gt; promote a function shift from oxidative cyclization to dehydrogenation.PMC12403149223227012022YFC3401500BNLMS-CXX-20210622193073CHE-215397292253305Yang JZhou QHouk KNDing QWu DGuo NWang QDong HFan JLei XGao LGu JLiu FfalseAspartic acid residues in BBE-like enzymes from &lt;i&gt;Morus alba&lt;/i&gt; promote a function shift from oxidative cyclization to dehydrogenation.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 <i>Morus alba</i> 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 Å and 2.21 Å 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 funct2025-01-01T00:00:00Z2025 Aug2026-07-16T05:35:33.222Z2026-07-09T10:39:15.958ZS-EPMC124031494082803010.1073/pnas.2504346122