Department of Chemistry and Biochemistry , University of Windsor , Windsor , Ontario N9B 3P4 , Canada.
J Phys Chem A. 2019 Sep 12;123(36):7710-7719. doi: 10.1021/acs.jpca.9b05914. Epub 2019 Sep 3.
Maleamate amidohydrolase (NicF) is a key enzyme in vitamin B metabolism that catalyzes the hydrolysis of maleamate to produce maleic acid and ammonia. Unlike most members from the amidohydrolase superfamily it does not require a metal ion. Here, we use multiscale computational enzymology to investigate the catalytic mechanism, substrate binding, oxyanion hole, and roles of key active site residues of NicF from . In particular, molecular dynamics (MD) simulations, quantum mechanics/molecular mechanics (QM/MM) and QTAIM methods have been applied. The mechanism of the NicF-catalyzed reaction proceeds by a nucleophilic addition-elimination sequence involving the formation of a thioester enzyme intermediate ( in stage 1) followed by hydrolysis of the thioester bond to form the products (stage 2). Consequently, the formation of in stage 1 is the rate-limiting step with a barrier of 88.8 kJ·mol relative to the reactant complex, . Comparisons with related metal-dependent enzymes, particularly the zinc-dependent nicotinamidase from (SpNic), have also been made to further illustrate unique features of the present mechanism. Along with -NH- donor groups of the oxyanion hole (i.e., HN-Thr146, HN-Cys150), the active site β-hydroxyl of threonine (HO-βThr146) is concluded to play a role in stabilizing the carbonyl oxygen of maleamate during the mechanism.
男性酰胺水解酶（NicF）是维生素 B 代谢中的一种关键酶，可催化男性酰胺水解生成马来酸和氨。与酰胺水解酶超家族的大多数成员不同，它不需要金属离子。在这里，我们使用多尺度计算酶学方法来研究 NicF 的催化机制、底物结合、氧阴离子空穴以及关键活性位点残基的作用。特别是，应用了分子动力学（MD）模拟、量子力学/分子力学（QM/MM）和 QTAIM 方法。NicF 催化反应的机制通过涉及形成硫酯酶中间物（在第 1 阶段）的亲核加成-消除序列进行，随后硫酯键水解形成产物（第 2 阶段）。因此，第 1 阶段中 的形成是限速步骤，相对于反应物复合物，其势垒为 88.8 kJ·mol。与相关的依赖金属的酶，特别是来自（SpNic）的锌依赖性烟酰胺酶进行了比较，进一步说明了本机制的独特特征。与氧阴离子空穴的 -NH-供体基团（即，HN-Thr146、HN-Cys150）一起，推测活性位点β-羟脯氨酸（HO-βThr146）在机制过程中在稳定男性酰胺的羰基氧方面起作用。
