Crystallography Coupled with Kinetic Analysis Provides Mechanistic Underpinnings of a Nicotine-Degrading Enzyme.
Crystallography Coupled with Kinetic Analysis Provides Mechanistic Underpinnings of a Nicotine-Degrading Enzyme.
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DOI：
10.1021/acs.biochem.8b00384
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发表时间：
2018-07-03
期刊：
影响因子：
2.9
通讯作者：
Allen KN
中科院分区：
文献类型：
--
作者：
Tararina MA;Xue S;Smith LC;Muellers SN;Miranda PO;Janda KD;Allen KN
尼古丁氧化还原酶（NicA 2）是一种细菌黄素酶，通过将S-尼古丁氧化为N-甲基麦斯明来催化尼古丁催化的第一步。由于其纳摩尔底物结合亲和力，它已被提议用作尼古丁成瘾的生物治疗剂。已经报道了NicA 2的第一个晶体结构，确立了NicA 2作为单胺氧化酶（MAO）家族的成员。然而，底物特异性和底物结合/催化的结构决定因素尚未探讨。在本文中，pH速率曲线、单转换动力学和结合数据的分析确定pH不显著影响催化速率，并且产物释放不是速率限制。X-射线晶体结构的NicA 2与S-尼古丁精制到2.65微米分辨率揭示了一个疏水结合位点与溶剂专用腔。疏水性相互作用主要定向的基板，促进结合的去质子化的物种和支持的去质子转移机制。值得注意的是，NicA 2对由人MAO的两种亚型氧化的神经递质没有显示出活性。为了进一步探测NicA 2的底物范围，使用一系列底物类似物评价酶活性，表明S-尼古丁是最佳底物，并且吡啶基环内的取代消除了NicA 2活性。此外，活性位点残基的诱变和动力学分析表明，去除S-尼古丁的吡啶环和T381的羟基之间的氢键对KM具有10倍的影响，支持这种键在定位底物的催化活性形式中的作用。总之，结晶学结合动力学分析提供了更深入的了解这种酶的显着特异性。
Nicotine oxidoreductase (NicA2) is a bacterial flavoenzyme, which catalyzes the first step of nicotine catabolism by oxidizing S-nicotine into N-methyl-myosmine. Its use has been proposed as a biotherapeutic for nicotine addiction due to its nanomolar substrate binding affinity. The first crystal structure of NicA2 has been reported, establishing NicA2 as a member of the monoamine oxidase (MAO) family. However, substrate specificity and structural determinants of substrate binding/catalysis have not been explored. Herein, analysis of pH-rate profile, single-turnover kinetics and binding data establish that pH does not significantly affect catalytic rate and product release is not rate limiting. The X-ray crystal structure of NicA2 with S-nicotine refined to 2.65 Å resolution reveals a hydrophobic binding site with a solvent exclusive cavity. Hydrophobic interactions predominantly orient the substrate, promoting the binding of a deprotonated species and supporting a hydride-transfer mechanism. Notably, NicA2 showed no activity against neurotransmitters oxidized by the two isoforms of human MAO. To further probe the substrate range of NicA2, enzyme activity was evaluated using a series of substrate analogs, indicating that S-nicotine is the optimal substrate and substitutions within the pyridyl ring abolish NicA2 activity. Moreover, mutagenesis and kinetic analysis of active-site residues reveal that removal of a hydrogen bond between the pyridyl ring of S-nicotine and the hydroxyl group of T381 has a 10-fold effect on KM, supporting the role of this bond in positioning the catalytically competent form of the substrate. Together, crystallography combined with kinetic analysis provide a deeper understanding of this enzyme’s remarkable specificity.
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