Structural and Functional Characteristics of Potent Dioxygenase from Moesziomyces aphidis

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, Moesziomyces aphidis DSM 70725 aromatic dioxygenase (MapADO) is characterized through X-ray crystallography, biophysical as well as biochemical assays, substrate docking and mutagenesis. MapADO features a seven-bladed β-propeller fold and a Fe2+ center coordinated by four histidine residues and shares a conserved structural motif with homologous enzymes despite low sequence identity (2+ is tightly bound and present in the catalytically active oxidation state at ambient conditions. MapADO is robust and retains activity for several freeze/thaw cycles. MapADO’s interaction with ligands 4-hydroxybenzaldehyde, ortho-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. MapADO wild-type exhibits the highest reported activity for converting isoeugenol to vanillin (231 μmol min–1 mg–1).酶促C=C双键断裂生成羰基类化合物，是替代臭氧分解或化学计量金属氧化剂使用的新兴策略。然而，四组氨酸配位铁双加氧酶（4-His Fe dioxygenases）的底物范围似乎仅局限于芳环4位带有羟基的芳香族化合物。深入的结构与功能表征，是理解并最终拓展这类酶家族底物范围的必要前提。 本研究通过X射线晶体学、生物物理及生化实验、底物分子对接与定点诱变技术，对蚜生掷孢酵母（Moesziomyces aphidis）DSM 70725来源的芳香族双加氧酶（MapADO）进行了系统表征。MapADO具有七叶β-折叠桶（seven-bladed β-propeller）折叠结构，以及由四个组氨酸残基配位的Fe²+活性中心；尽管序列同源性较低，但其仍与同源酶共享保守的结构基序，且Fe²+在常温条件下紧密结合，并以催化活性氧化态存在。 MapADO稳定性优异，历经多次冻融循环仍可保留催化活性。MapADO与配体4-羟基苯甲醛、邻香草醛及香草醛的相互作用研究表明，酚羟基的氢键作用是其发挥催化活性的关键。结构分析与定点诱变实验表明，两个关键残基Y136与K169，以及底物的羟基基团，对于将双键精准定位至Fe中心的活化氧位点至关重要。野生型MapADO在将异丁香酚转化为香草醛的反应中，展现出目前已报道的最高催化活性（231 μmol·min⁻¹·mg⁻¹）。