Exploiting correlated molecular-dynamics networks to counteract enzyme activity–stability trade-off

Yu, Haoran; Dalby, Paul A.

doi:10.1073/pnas.1812204115

Cited by 77 publications

(75 citation statements)

References 28 publications

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“…For enzymes in particular, previous studies have shown that missense mutations often act pleiotropically in which catalytically enhancing mutations are, on average, moderately destabilizing (6,(10)(11). Consequently, high basal stability can buffer catalytically beneficial but destabilizing mutations (12)(13), allowing fixation.…”

Section: Introductionmentioning

confidence: 99%

Impact of in vivo protein folding probability on local fitness landscapes

Faber

Wrenbeck

Azouz

et al. 2019

Preprint

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It is incompletely understood how biophysical properties like protein stability impact molecular evolution and epistasis. Epistasis is defined as specific when a mutation exclusively influences the phenotypic effect of another mutation, often at physically interacting residues. By contrast, nonspecific epistasis results when a mutation is influenced by a large number of non-local mutations. As most mutations are pleiotropic, basal protein stability is thought to determine activity-enhancing mutational tolerance, which implies that nonspecific epistasis is dominant. However, evidence exists for both specific and nonspecific epistasis as the prevalent factor, with limited comprehensive datasets to validate either claim. Here we use deep mutational scanning to probe how initial enzyme stability impacts local fitness landscapes. We computationally designed two different variants of the amidase AmiE in which catalytic efficiencies are statistically indistinguishable but the enzyme variants have lower probabilities of folding in vivo.Local fitness landscapes show only slight alterations among variants, with essentially the same global distribution of fitness effects. However, specific epistasis was predominant for the subset of mutations exhibiting positive sign epistasis. These mutations mapped to spatially distinct locations on AmiE near the initial mutation or proximal to the active site. Most intriguingly, the majority of specific epistatic mutations were codondependent, with different synonymous codons resulting in fitness sign reversals.Together, these results offer a nuanced view of how protein stability impacts local fitness landscapes, and suggest that transcriptional-translational effects are an equally important determinant as thermodynamic stability in determining evolutionary outcomes.

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Section: Introductionmentioning

confidence: 99%

Impact of in vivo protein folding probability on local fitness landscapes

Faber

Wrenbeck

Azouz

et al. 2019

Preprint

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“…Therefore, we used a previously stabilised variant H192P/A282P/I365L/G506A (4M) as the template for constructing the new library to promote evolvability . Introduction of these same stabilising mutations was recently found to restore the stability loss of another TK variant (S385D/D469T/R520Q) that was engineered to accepted aromatic aldehydes .…”

Section: Resultsmentioning

confidence: 99%

Engineering transketolase to accept both unnatural donor and acceptor substrates and produce α‐hydroxyketones

López

Steadman

et al. 2019

The FEBS Journal

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A narrow substrate range is a major limitation in exploiting enzymes more widely as catalysts in synthetic organic chemistry. For enzymes using two substrates, the simultaneous optimisation of both substrate specificities is also required for the rapid expansion of accepted substrates. Transketolase (TK) catalyses the reversible transfer of a C 2 -ketol unit from a donor substrate to an aldehyde acceptor and suffers the limitation of narrow substrate scope for industrial applications. Herein, TK from Escherichia coli was engineered to accept both pyruvate, as a novel donor substrate, and unnatural acceptor aldehydes, including propanal, pentanal, hexanal and 3-formylbenzoic acid (FBA). Twenty single-mutant variants were first designed and characterised experimentally. Beneficial mutations were then recombined to construct a small library. Screening of this library identified the best variant with a 9.2-fold improvement in the yield towards pyruvate and propionaldehyde, relative to wild-type (WT). Pentanal and hexanal were used as acceptors to determine stereoselectivities of the reactions, which were found to be higher than 98% enantiomeric excess (ee) for the S configuration. Three variants were identified to be active for the reaction between pyruvate and 3-FBA. The best variant was able to convert 47% of substrate into product within 24 h, whereas no conversion was observed for WT. Docking experiments suggested a cooperation between the mutations responsible for donor and acceptor recognition, which would promote the activity towards both the acceptor and donor. The variants obtained have the potential to be used for developing catalytic pathways to a diverse range of high-value products.

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“…Thermal stability and enzyme dynamics : Dalby and coworkers recently described their experience of epistatic (non‐additive) effects from mutations located in distal parts of E. coli transketolase (TK) while attempting to improve its thermal stability . Starting from the wild‐type enzyme, the investigators created several TK variants with single, double, triple and quadruple mutations.…”

Section: Evolving and Engineering Enzymes Based On Dynamicsmentioning

confidence: 99%