A Single-Point Mutation in d-Arginine Dehydrogenase Unlocks a Transient Conformational State Resulting in Altered Cofactor Reactivity

Abstract

Proteins are inherently dynamic, and proper enzyme function relies on conformational flexibility. In this study, we demonstrated how an active site residue changes an enzyme’s reactivity by modulating fluctuations between conformational states. Replacement of tyrosine 249 (Y249) with phenylalanine in the active site of the flavin-dependent d-arginine dehydrogenase yielded an enzyme with both an active yellow FAD (Y249F-y) and an inactive chemically modified green FAD, identified as 6-OH-FAD (Y249F-g) through various spectroscopic techniques. Structural investigation of Y249F-g and Y249F-y variants by comparison to the wild-type enzyme showed no differences in the overall protein structure and fold. A closer observation of the active site of the Y249F-y enzyme revealed an alternative conformation for some active site residues and the flavin cofactor. Molecular dynamics simulations probed the alternate conformations observed in the Y249F-y enzyme structure and showed that the enzyme variant with FAD samples a metastable conformational state, not available to the wild-type enzyme. Hybrid quantum/molecular mechanical calculations identified differences in flavin electronics between the wild type and the alternate conformation of the Y249F-y enzyme. The computational studies further indicated that the alternate conformation in the Y249F-y enzyme is responsible for the higher spin density at the C6 atom of flavin, which is consistent with the formation of 6-OH-FAD in the variant enzyme. The observations in this study are consistent with an alternate conformational space that results in fine-tuning the microenvironment around a versatile cofactor playing a critical role in enzyme function.

Quantum‐based Modeling of Protein‐ligand Interaction: The Complex of RutA with Uracil and Molecular Oxygen

“…Figure 4d illustrates the consequences of such modeling showing that the covalently bound adduct is formed in less than 1 ps after spontaneous binding oxygen to the C6 atom of flavin. The C6-peroxo species (Figure 4d) is a possible precursor of the C6OH-flavin derivative; the recent QM/MM simulations [24] suggest that the interaction with the protein increase spin Analyzing these results, we observe significant charge transfer to the oxygen molecule at many steps of the simulation. Such structures are characterized by: (i) close distances of oxygen to the flavin, (ii) close position of the Lys69 sidechain, (iii) proper position of the uracil that allows for hydrogen bonding network to stabilize the charge transfer.…”

Protein Conformational Space at the Edge of Allostery: Turning a Nonallosteric Malate Dehydrogenase into an “Allosterized” Enzyme Using Evolution-Guided Punctual Mutations

“…This is because, in enzymes, the dynamical process is hierarchical and covers different time scales that influence functionality and allostery ( Frauenfelder et al 2001 ; Wrab et al 2011 ; Choy et al 2017 ; Fleishman and Horovitz 2021 ; Wolf et al 2021 ; Madhu et al 2022 ; Makurat et al 2022 ). Because of this hierarchy, very short time scale dynamics, due to simple side-chain fluctuations of a single residue can influence reorganization between the T- and R-states ( Zhou et al 2003 ; Fujiwara et al 2017 ; Iyer et al 2021 ). How the enzymatic activation (heterotropic activation) of LDHs by the allosteric effector (FBP) evolved (concomitantly or successively) requires additional investigations.…”

An Alternative Strategy for Spectral Tuning of Flavin-binding Fluorescent Proteins

“…(61) We recently developed ASEC-FEG for flavoproteins. (56,62) With ASEC, the quantum chemical calculations are performed in the field of a time-averaged electrostatic potential environment of the protein and solution (collectively referred to as a "solvent" in the ASEC acronym). Effectively, the protein and solution are represented as a "superposition" of structures obtained from MD simulations.…”