Department of Chemistry, Yale University, 225 Prospect Street, New Haven, Connecticut 06520, United States.
Biochemistry. 2020 Mar 3;59(8):943-954. doi: 10.1021/acs.biochem.9b01089. Epub 2020 Feb 14.
The projected decline of available phosphorus necessitates alternative methods to derive usable phosphate for fertilizer and other applications. Phosphite dehydrogenase oxidizes phosphite to phosphate with the cofactor NAD serving as the hydride acceptor. In addition to producing phosphate, this enzyme plays an important role in NADH cofactor regeneration processes. Mixed quantum mechanical/molecular mechanical free energy simulations were performed to elucidate the mechanism of this enzyme and to identify the protonation states of the substrate and product. Specifically, the finite temperature string method with umbrella sampling was used to generate the free energy surfaces and determine the minimum free energy paths for six different initial conditions that varied in the protonation state of the substrate and the position of the nucleophilic water molecule. In contrast to previous studies, the mechanism predicted by all six independent strings is a concerted but asynchronous dissociative mechanism in which hydride transfer from the phosphite substrate to NAD occurs prior to attack by the nucleophilic water molecule. His292 is identified as the most likely general base that deprotonates the attacking water molecule. However, Arg237 could also serve as this base if it were deprotonated and His292 were protonated prior to the main chemical transformation, although this scenario is less probable. The simulations indicate that the phosphite substrate is monoanionic in its active form and that the most likely product is dihydrogen phosphate. These mechanistic insights may be helpful for designing mutant enzymes or artificial constructs that convert phosphite to phosphate and NAD to NADH more effectively.
预计可用磷的减少需要替代方法来获得可用于肥料和其他应用的磷酸盐。亚磷酸盐脱氢酶将亚磷酸盐氧化为磷酸盐，辅酶 NAD 作为氢化物受体。除了产生磷酸盐外，该酶在 NADH 辅酶再生过程中也起着重要作用。进行了混合量子力学/分子力学自由能模拟，以阐明该酶的机制并确定底物和产物的质子化状态。具体来说，使用有限温度弦法和伞状采样生成了自由能表面，并确定了六个不同初始条件的最小自由能路径，这些初始条件在底物的质子化状态和亲核水分子的位置上有所不同。与先前的研究相比，所有六个独立弦预测的机制是一个协同但不同步的解离机制，其中亚磷酸盐底物的氢化物转移先于亲核水分子的攻击。His292 被确定为最有可能使攻击水分子去质子化的通用碱。然而，如果 Arg237 在主要化学转化之前去质子化并且 His292 被质子化，它也可以作为该碱，尽管这种情况不太可能发生。模拟表明，在其活性形式中，亚磷酸盐底物是单阴离子，最可能的产物是磷酸二氢盐。这些机制上的见解可能有助于设计突变酶或人工构建体，以更有效地将亚磷酸盐转化为磷酸盐和 NAD 转化为 NADH。
