Increased Dynamic Effects in a Catalytically Compromised Variant of<i>Escherichia coli</i>Dihydrofolate Reductase

Ruiz‐Pernía, J. Javier; Luk, Louis Y. P.; García-Meseguer, Rafael; Martí, Sérgio; Loveridge, E. Joel; Tuñón, Iñaki; Moliner, Vicent; Allemann, Rudolf Konrad

doi:10.1021/ja410519h

Cited by 56 publications

(146 citation statements)

References 52 publications

Supporting

Mentioning

130

Contrasting

Unclassified

Order By: Relevance

“…The temperature dependences of the tunneling contributions ( κ ) are identical in the light and heavy enzyme for both benzyl alcohol and isopropanol (Tables S4, S5). This observation is in agreement with our previous computational and experimental studies, which indicated that tunneling or barrier modulation were not driven by compressive “promoting motions” 2, 3c, 11…”

supporting

confidence: 93%

Isotope Substitution of Promiscuous Alcohol Dehydrogenase Reveals the Origin of Substrate Preference in the Transition State

et al. 2018

Self Cite

View full text Add to dashboard Cite

The origin of substrate preference in promiscuous enzymes was investigated by enzyme isotope labelling of the alcohol dehydrogenase from Geobacillus stearothermophilus (BsADH). At physiological temperature, protein dynamic coupling to the reaction coordinate was insignificant. However, the extent of dynamic coupling was highly substrate‐dependent at lower temperatures. For benzyl alcohol, an enzyme isotope effect larger than unity was observed, whereas the enzyme isotope effect was close to unity for isopropanol. Frequency motion analysis on the transition states revealed that residues surrounding the active site undergo substantial displacement during catalysis for sterically bulky alcohols. BsADH prefers smaller substrates, which cause less protein friction along the reaction coordinate and reduced frequencies of dynamic recrossing. This hypothesis allows a prediction of the trend of enzyme isotope effects for a wide variety of substrates.

show abstract

supporting

confidence: 93%

Isotope Substitution of Promiscuous Alcohol Dehydrogenase Reveals the Origin of Substrate Preference in the Transition State

et al. 2018

Self Cite

View full text Add to dashboard Cite

show abstract

“…However, we note that, in a contrasting view, based on extensive experimental work involving protein labelling studies, Alleman and co-workers have argued that dynamic coupling is in fact detrimental to catalysis by DHFR, and by the mesophile EcDHFR [55]. By comparing DHFRs from organisms that have adapted to survive at a wide range of temperatures, the authors concluded that the dynamical coupling has been minimized during evolution and is rather a consequence of reorganizational motions that are necessary to facilitate charge transfer effects.…”

Section: Dihydrofolate Reductasementioning

confidence: 73%

Conformational dynamics and enzyme evolution

Petrović

Risso

Kamerlin

et al. 2018

J. R. Soc. Interface.

172

184

View full text Add to dashboard Cite

Enzymes are dynamic entities, and their dynamic properties are clearly linked to their biological function. It follows that dynamics ought to play an essential role in enzyme evolution. Indeed, a link between conformational diversity and the emergence of new enzyme functionalities has been recognized for many years. However, it is only recently that state-ofthe-art computational and experimental approaches are revealing the crucial molecular details of this link. Specifically, evolutionary trajectories leading to functional optimization for a given host environment or to the emergence of a new function typically involve enriching catalytically competent conformations and/or the freezing out of non-competent conformations of an enzyme. In some cases, these evolutionary changes are achieved through distant mutations that shift the protein ensemble towards productive conformations. Multifunctional intermediates in evolutionary trajectories are probably multi-conformational, i.e. able to switch between different overall conformations, each competent for a given function. Conformational diversity can assist the emergence of a completely new active site through a single mutation by facilitating transition-state binding. We propose that this mechanism may have played a role in the emergence of enzymes at the primordial, progenote stage, where it was plausibly promoted by high environmental temperatures and the possibility of additional phenotypic mutations.

show abstract

“…In enzymes where vibrations lead to active site 'compression' such as the transient reduction in hydrogen donor-acceptor atoms, which has been suggested for OYEs such as PETNR, 1,25,[39][40][41] then perturbation of such vibrations can lead to altered reaction kinetics by altering the probability of transition state formation and/or the rate of transition state recrossing. 17,18,42 Slower dynamics will also be affected by mass perturbation, which may lead to altered substrate/product binding and release kinetics. 20,21 We have previously identified computationally specific vibrational modes that reduce the donor (NADH C4)-acceptor (FMN N5) distance in the PETNR homolog morphinone reductase.…”

Section: Resultsmentioning

confidence: 99%

Untangling Heavy Protein and Cofactor Isotope Effects on Enzyme-Catalyzed Hydride Transfer

et al. 2016

View full text Add to dashboard Cite

'Heavy' (isotopically-labeled) enzyme isotope effects offer a direct experimental probe of the role of protein vibrations on enzyme-catalyzed reactions. Here we have developed a strategy to generate isotopologues of the flavoenzyme pentaerythritol tetranitrate reductase (PETNR) where the protein and/or intrinsic flavin mononucleotide (FMN) cofactor are isotopically labeled with 2 H, 15 N and 13 C. Both the protein and cofactor contribute to the enzyme isotope effect on the reductive hydride transfer reaction, but their contributions are not additive and may partially cancel each other out. However, the isotope effect specifically arising from the FMN suggests that vibrations local to the active site play a role in the hydride transfer chemistry, while the protein-only 'heavy enzyme' effect demonstrates that protein vibrations contribute to catalysis in PETNR. In all cases, enthalpy-entropy compensation plays a major role in minimizing the magnitude of 'heavy enzyme' isotope effects. Fluorescence lifetime measurements of the intrinsic flavin mononucleotide show marked differences between 'light' and 'heavy' enzymes on the ns-ps timescale, suggesting relevant timescale(s) for those vibrations implicated in the 'heavy enzyme' isotope effect on the PETNR reaction.

show abstract

Increased Dynamic Effects in a Catalytically Compromised Variant ofEscherichia coliDihydrofolate Reductase

Cited by 56 publications

References 52 publications

Isotope Substitution of Promiscuous Alcohol Dehydrogenase Reveals the Origin of Substrate Preference in the Transition State

Isotope Substitution of Promiscuous Alcohol Dehydrogenase Reveals the Origin of Substrate Preference in the Transition State

Conformational dynamics and enzyme evolution

Untangling Heavy Protein and Cofactor Isotope Effects on Enzyme-Catalyzed Hydride Transfer

Contact Info

Product

Resources

About