Schober Lukas, Plewka Jacek, Sriwaiyaphram Kanokkan, Bielec Björn, Schiefer Astrid, Wongnate Thanyaporn, Magiera-Mularz Katarzyna, Rudroff Florian, Winkler Margit
Institute of Molecular Biotechnology, Graz University of Technology, NAWI Graz, Petersgasse 14, 8010 Graz, Austria.
Department of Organic Chemistry, Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Krakow, Poland.
JACS Au. 2025 Jun 12;5(7):3014-3020. doi: 10.1021/jacsau.5c00456. eCollection 2025 Jul 28.
Enzymatic C=C double bond cleavage to give carbonyl-species is an emerging alternative to ozonolysis, or stoichiometric use of metal-oxidants. The substrate scope of 4-His Fe dioxygenases, however, appears to be restricted to aromatic compounds with a hydroxy group at the 4-position of the aromatic ring. In-depth structural and functional characterization is a prerequisite to understand and ultimately to extend the substrate scope of this family of enzymes. Herein, DSM 70725 aromatic dioxygenase (ADO) is characterized through X-ray crystallography, biophysical as well as biochemical assays, substrate docking and mutagenesis. ADO features a seven-bladed β-propeller fold and a Fe center coordinated by four histidine residues and shares a conserved structural motif with homologous enzymes despite low sequence identity (<38%). Fe is tightly bound and present in the catalytically active oxidation state at ambient conditions. ADO is robust and retains activity for several freeze/thaw cycles. ADO's interaction with ligands 4-hydroxybenzaldehyde, -vanillin and vanillin indicate that hydrogen-bonding of the phenolic OH group is key to activity. Structural analysis and site-directed mutagenesis indicate that two key residues (Y136 and K169), and the substrate's hydroxy group, are essential for accurately positioning the double bond toward the activated oxygen at the Fe center. ADO wild-type exhibits the highest reported activity for converting isoeugenol to vanillin (231 μmol min mg).
酶促C=C双键断裂生成羰基化合物是一种新兴的替代臭氧分解或化学计量使用金属氧化剂的方法。然而，4-组氨酸铁双加氧酶的底物范围似乎仅限于在芳环4位带有羟基的芳香族化合物。深入的结构和功能表征是理解并最终扩展该酶家族底物范围的先决条件。在此，通过X射线晶体学、生物物理以及生化分析、底物对接和诱变对DSM 70725芳香族双加氧酶（ADO）进行了表征。ADO具有七叶β-螺旋桨折叠结构和由四个组氨酸残基配位的铁中心，尽管序列同一性较低（<38%），但与同源酶共享一个保守的结构基序。铁紧密结合并在环境条件下以催化活性氧化态存在。ADO很稳定，在几个冻融循环后仍保持活性。ADO与配体4-羟基苯甲醛、香草醛和香兰素的相互作用表明，酚羟基的氢键作用是活性的关键。结构分析和定点诱变表明，两个关键残基（Y136和K169）以及底物的羟基对于将双键准确地定位到铁中心的活化氧至关重要。ADO野生型在将异丁香酚转化为香草醛方面表现出最高的报道活性（231 μmol min mg）。
