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Minimization of dynamic effects in the evolution of dihydrofolate reductase.

ABSTRACT: Protein isotope labeling is a powerful technique to probe functionally important motions in enzyme catalysis and can be applied to investigate the conformational dynamics of proteins. Previous investigations have indicated that dynamic coupling is detrimental to catalysis by dihydrofolate reductase (DHFR) from the mesophile Escherichia coli (EcDHFR). Comparison of DHFRs from organisms adapted to survive at a wide range of temperatures suggests that dynamic coupling in DHFR catalysis has been minimized during evolution; it arises from reorganizational motions needed to facilitate charge transfer events. Contrary to the behaviour observed for the DHFR from the moderate thermophile Geobacillus stearothermophilus (BsDHFR), the chemical transformation catalyzed by the cold-adapted bacterium Moritella profunda (MpDHFR) is only weakly affected by protein isotope substitutions at low temperatures, but the isotopically substituted enzyme is a substantially inferior catalyst at higher, non-physiological temperatures. QM/MM studies revealed that this behaviour is caused by the enzyme's structural sensitivity to temperature changes, which enhances unfavorable dynamic coupling at higher temperatures by promoting additional recrossing trajectories on the transition state dividing surface. We postulate that these motions are minimized by fine-tuning DHFR flexibility through optimization of the free energy surface of the reaction, such that a nearly static reaction-ready configuration with optimal electrostatic properties is maintained under physiological conditions.

SUBMITTER: Ruiz-Pernia JJ

PROVIDER: S-EPMC6006479 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Minimization of dynamic effects in the evolution of dihydrofolate reductase.

Ruiz-Pernía J Javier JJ Behiry Enas E Luk Louis Y P LYP Loveridge E Joel EJ Tuñón Iñaki I Moliner Vicent V Allemann Rudolf K RK

Chemical science 20160203 5

Protein isotope labeling is a powerful technique to probe functionally important motions in enzyme catalysis and can be applied to investigate the conformational dynamics of proteins. Previous investigations have indicated that dynamic coupling is detrimental to catalysis by dihydrofolate reductase (DHFR) from the mesophile <i>Escherichia coli</i> (EcDHFR). Comparison of DHFRs from organisms adapted to survive at a wide range of temperatures suggests that dynamic coupling in DHFR catalysis has b ...[more]

PMID: 29997817

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Project description:Correlated networks of amino acids have been proposed to play a fundamental role in allostery and enzyme catalysis. These networks of amino acids can be traced from surface-exposed residues all the way into the active site, and disruption of these networks can decrease enzyme activity. Substitution of the distal Gly121 residue in Escherichia coli dihydrofolate reductase results in an up to 200-fold decrease in the hydride transfer rate despite the fact that the residue is located 15 Å from the active-site center. In this study, nuclear magnetic resonance relaxation experiments are used to demonstrate that dynamics on the picosecond to nanosecond and microsecond to millisecond time scales are changed significantly in the G121V mutant of dihydrofolate reductase. In particular, picosecond to nanosecond time scale dynamics are decreased in the FG loop (containing the mutated residue at position 121) and the neighboring active-site loop (the Met20 loop) in the mutant compared to those of the wild-type enzyme, suggesting that these loops are dynamically coupled. Changes in methyl order parameters reveal a pathway by which dynamic perturbations can be propagated more than 25 Å across the protein from the site of mutation. All of the enzyme complexes, including the model Michaelis complex with folate and nicotinamide adenine dinucleotide phosphate bound, assume an occluded ground-state conformation, and we do not observe sampling of a higher-energy closed conformation by (15)N R2 relaxation dispersion experiments. This is highly significant, because it is only in the closed conformation that the cofactor and substrate reactive centers are positioned for reaction. The mutation also impairs microsecond to millisecond time scale fluctuations that have been implicated in the release of product from the wild-type enzyme. Our results are consistent with an important role for Gly121 in controlling protein dynamics critical for enzyme function and further validate the dynamic energy landscape hypothesis of enzyme catalysis.

Dataset Information

Minimization of dynamic effects in the evolution of dihydrofolate reductase.

Publications

Minimization of dynamic effects in the evolution of dihydrofolate reductase.

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