bi9b01089_si_001.pdf (3.72 MB)
Examining the Mechanism of Phosphite Dehydrogenase with Quantum Mechanical/Molecular Mechanical Free Energy Simulations
journal contribution
posted on 2020-02-13, 22:43 authored by David
R. Stevens, Sharon Hammes-SchifferThe
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.