Origin of Stereoselectivity and Substrate/Ligand Recognition in an FAD-Dependent R-Selective Amine Oxidase
Origin of Stereoselectivity and Substrate/Ligand Recognition in an FAD-Dependent R-Selective Amine Oxidase
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DOI：
10.1021/acs.jpcb.6b09328
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发表时间：
2016-10-20
影响因子：
3.3
通讯作者：
Asano, Yasuhisa
中科院分区：
文献类型：
--
作者：
Nakano, Shogo;Yasukawa, Kazuyuki;Asano, Yasuhisa
阐明胺氧化酶(AOX)的分子机理有助于通过合理的设计来扩大它们的反应活性，并将其应用于各种胺类化合物的去消旋反应。到目前为止，已经对S选择性AOX进行了几项研究，但相对较少的研究集中在R选择性AOX上。在本研究中，我们试图通过将Y228L和R283G突变引入猪肾脏的D-氨基酸氧化酶来阐明pkAOx的作用机制。底物结合蛋白的四种晶体结构和基于关联碎片分子轨道(FMO)的第一性原理计算表明，两个芳香族残基Tyr224和Phe242与底物形成稳定的pi-pi堆积作用。酶动力学也支持Tyr224在催化中的重要性：当以(R)-甲基苄胺[(R)-MBA]或(R)-1-(2-萘基)乙胺[(R)-NEA]为底物时，Y224L突变体的k(Cat)/K-m值比野生型(WT)降低了300倍。另一方面，几个Phe242突变体对(R)-NEA的反应活性高于WT酶。此外，FMO分析表明，pkAOx与(R)-MBA的相互作用比与(S)-MBA更稳定，这种能量差异有助于(R)-MBA在外消旋体中的特异性识别。通过本研究，我们阐明了pkAOx的三个特征：Tyr224和Phe242在催化中的作用，高立体选择性的来源，以及扩大其对含大基胺类化合物的反应能力的可能性。
Elucidation of the molecular mechanism of amine oxidases (AOx) will help to extend their reactivity by rational design and their application to deracemization of various amine compounds. To date, several studies have been performed on S-selective AOx, but relatively few have focused on R-selective AOx. In this study, we sought to elucidate the mechanism of pkAOx, an R-selective AOx that we designed by introducing the Y228L and R283G mutations into D-amino acid oxidase from pig kidney. Four crystal structures of the substrate-bound protein and first-principles calculations based on the correlated fragment molecular orbital (FMO) indicated that two aromatic residues, Tyr224 and Phe242, form stable pi-pi stacking interaction with substrates. Enzyme kinetics also supported the importance of Tyr224 in catalysis: the k(cat)/K-m value of the Y224L mutant was reduced by 300-fold than that of wild-type (WT) when utilizing either (R)-methylbenzylamine [(R)-MBA] or (R)-1-(2-naphthyl)ethylamine [(R)-NEA] as the substrate. On the other hand, several Phe242 mutants exhibited higher reactivity toward (R)-NEA than the WT enzyme. In addition, FMO analysis indicated that pkAOx forms similar to 13 kcal/mol more stable interaction with (R)-MBA than with (S)-MBA; this energy difference contributes to specific recognition of (R)-MBA in the racemate. Through the present study, we clarified three features of pkAOx: the roles of Tyr224 and Phe242 in catalysis, the origin of high stereoselectivity, and the potential to extend its reactivity toward amine compounds with bulky groups.