Mechanisms of Enzymatic Vinyl Isonitrile Formation and Reprogramming of Isonitrilases and Iron/2-Oxoglutarate Desaturases

酶促乙烯基异腈形成的机制以及异腈酶和铁/2-氧戊二酸去饱和酶的重编程

• 批准号: 10402382
• 负责人: Wei-chen Chang
• 金额: $ 39.04万
• 依托单位: NORTH CAROLINA STATE UNIVERSITY RALEIGH
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10402382
• 关键词: Address; Alkenes; Amines; Amino Acid Sequence; Amino Acid Substitution; Anti-Bacterial Agents; Antibiotics; Biological; Biological Assay; Biomimetics; Biophysics; Carbohydrates; Carbon; Cells; Chemicals; Coupled; Coupling; Cyclization; DNA; Decarboxylation; Development; Directed Molecular Evolution; Electron Spin Resonance Spectroscopy; Engineering; Enzymes; Equation; Event; Genomics; Glutarates; Goals; Homologous Gene; Hydroxylation; In Vitro; Indoles; Iron; Isotope Labeling; Kinetics; Libraries; Logic; Mixed Function Oxygenases; Molecular; Mossbauer Spectroscopy; Natural Products; Nature; Nitriles; Organism; Outcome; Oxygenases; Pathway interactions; Positioning Attribute; Production; Property; Proteins; Publishing; Reaction; Sampling; Spectrometry; Succinates; Synthesis Chemistry; System; Testing; Tryptophan; Tyrosine; Variant; alpha ketoglutarate; analog; anti-cancer; base; carbonyl group; dehydrogenation; deprotonation; desaturase; design; enzyme mechanism; feeding; flexibility; functional group; halogenation; improved; in vitro activity; inorganic phosphate; mutant; reconstitution; ribulose 5-phosphate; screening

Project Summary/Abstract Isonitrilase is involved in constructing a triple bond from a primary amine and a carbonyl moiety of carbohydrate phosphate. Non-heme iron and 2-(oxo)glutarate-dependent (Fe/2OG) enzymes catalyze a bewildering array of transformations, which include halogenations, cyclizations, dehydrogenations, endoperoxidation and stereoinversion of aliphatic carbon centers. In more than 40 isolated vinyl isonitrile containing natural products, a universal approach involving a consecutive two-step enzymatic transformation by an isonitrilase and an Fe/2OG dependent alkene forming enzyme is proposed to install these functional groups. However, the mechanistic understating of these transformations remains to be elucidated. A chemical and biosynthetic understanding of these reaction mechanisms could enable reprograming of isonitrilase and Fe/2OG enzymes as biocatalysts for producing new compounds with improved bioactivity. Having recently made progress toward Fe/2OG enzyme catalyzed epoxidation and desaturation, and isonitrilase activity reconstitution in vitro, we propose to study the isonitrile and decarboxylation-assisted alkene formation reactions on the pathway to rhabduscin and paerucumarin biosyntheses. We will use an integrated approach to provide molecular understanding of the enzyme mechanisms and, in select cases, evaluate the substrate flexibility and reprogram the related enzymes by directed evolution.

项目总结/摘要 异腈水解酶参与从伯胺和碳水化合物的羰基部分构建三键 磷酸盐非血红素铁和2-（氧代）戊二酸依赖性（Fe/2 OG）酶催化一系列令人困惑的铁代谢。 转化，其包括卤化、环化、双化、内过氧化和 脂肪族碳中心的立体转化。在40多种分离的含乙烯基异腈的天然产物中， 一种通用的方法，包括通过异腈水解酶的连续两步酶促转化和 Fe/2 OG依赖性烯烃形成酶被提出来安装这些官能团。但 对这些转变的机械性理解仍有待阐明。化学和生物合成 了解这些反应机制可以使异腈水解酶和Fe/2 OG酶重新编程 作为生物催化剂用于生产具有改进的生物活性的新化合物。最近取得了进展， Fe/2 OG酶催化环氧化和去饱和反应，以及体外异腈酶活性重建， 建议研究异腈和脱羧辅助烯烃形成反应的途径， rhabduscin和paerucumarin生物合成。我们将使用综合方法提供分子 了解酶的机制，并在选择的情况下，评估底物的灵活性和重编程 相关酶的定向进化。

项目成果

Elucidating the Reaction Pathway of Decarboxylation-Assisted Olefination Catalyzed by a Mononuclear Non-Heme Iron Enzyme.

阐明由单核非血红素铁酶催化的脱羧辅助烯烃的反应途径。

• DOI: 10.1021/jacs.8b10077
• 发表时间: 2018-11-14
• 期刊: Journal of the American Chemical Society
• 影响因子: 15
• 作者: Yu CP;Tang Y;Cha L;Milikisiyants S;Smirnova TI;Smirnov AI;Guo Y;Chang WC
• 通讯作者: Chang WC

Current Understanding toward Isonitrile Group Biosynthesis and Mechanism.

• DOI: 10.1002/cjoc.202000448
• 发表时间: 2021-03
• 期刊: Chinese journal of chemistry
• 影响因子: 5.4
• 作者: Chen TY;Chen J;Tang Y;Zhou J;Guo Y;Chang WC
• 通讯作者: Chang WC

Identification of Cyclopropane Formation in the Biosyntheses of Hormaomycins and Belactosins: Sequential Nitration and Cyclopropanation by Metalloenzymes.

• DOI: 10.1002/anie.202113189
• 发表时间: 2022-02-07
• 期刊: Angewandte Chemie (International ed. in English)
• 作者: Li X;Shimaya R;Dairi T;Chang WC;Ogasawara Y
• 通讯作者: Ogasawara Y

BesC Initiates C-C Cleavage through a Substrate-Triggered and Reactive Diferric-Peroxo Intermediate.

• DOI: 10.1021/jacs.1c11109
• 发表时间: 2021-12-22
• 期刊: Journal of the American Chemical Society
• 影响因子: 15
• 作者: Manley OM;Tang H;Xue S;Guo Y;Chang WC;Makris TM
• 通讯作者: Makris TM

Repurposing Iron‐ and 2‐Oxoglutarate‐Dependent Oxygenases to Catalyze Olefin Hydration

重新利用铁和 2-氧戊二酸-依赖性氧化酶来催化烯烃水合

• DOI: 10.1002/anie.202311099
• 发表时间: 2023
• 期刊: Angewandte Chemie International Edition
• 作者: Wang, Bingnan;Lu, Yong;Cha, Lide;Chen, Tzu‐Yu;Palacios, Philip M.;Li, Liping;Guo, Yisong;Chang, Wei‐chen;Chen, Chuo
• 通讯作者: Chen, Chuo