Examining the Mechanism of Phosphite Dehydrogenase with Quantum Mechanical/Molecular Mechanical Free Energy Simulations

Abstract

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.

Serial crystallography captures dynamic control of sequential electron and proton transfer events in a flavoenzyme

“…Thirdly, despite its high sidechain pKa, the function of arginine as a proton donor has been suggested for several enzymes [52][53][54][55][56] . Our results provide structural intermediates before and after protonation of isoalloxazine N5 by Arg378 in the MmCPDII photolyase.…”

Multiple modes of substrate hydrolysis-induced covalent modification of strigolactone receptors

“…The string method is a transition path search method a path is discretized into images and the images are updated iteratively using restrained simulations [18][19][20] . This method has been previously used in conjunction with QM/MM simulations to study other enzymatic systems [21][22][23][24] .…”

“…The string method is a transition path search method a path is discretized into images and the images are updated iteratively using restrained simulations 18–20 . This method has been previously used in conjunction with QM/MM simulations to study other enzymatic systems 21–24 . String method optimization additionally enables computation of free energy profiles along reaction pathways, which in turn enables identification of the most probable of a set of possible reaction pathways.…”