Extreme Synergistic Mutational Effects in the Directed Evolution of a Baeyer–Villiger Monooxygenase as Catalyst for Asymmetric Sulfoxidation

Zhang, Zhigang; Londsdale, Richard; Sanchis, Joaquı́n; Reetz, Manfred T.

doi:10.1021/ja5098034

Cited by 70 publications

(84 citation statements)

References 85 publications

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“…PTE, a phosphotriesterase, was evolved toward arylesterase activity, and then back to its original PTE activity . The last two studies represent the rational design of enzymes in the laboratory, through the improvement of enantioselectivity in ANEH epoxide hydrolase and PAMO Baeyer−Villiger monooxygenase towards a thioether …”

Section: Resultsmentioning

confidence: 99%

How mutational epistasis impairs predictability in protein evolution and design

2016

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There has been much debate about the extent to which mutational epistasis, that is, the dependence of the outcome of a mutation on the genetic background, constrains evolutionary trajectories. The degree of unpredictability introduced by epistasis, due to the non-additivity of functional effects, strongly hinders the strategies developed in protein design and engineering. While many studies have addressed this issue through systematic characterization of evolutionary trajectories within individual enzymes, the field lacks a consensus view on this matter. In this work, we performed a comprehensive analysis of epistasis by analyzing the mutational effects from nine adaptive trajectories toward new enzymatic functions. We quantified epistasis by comparing the effect of mutations occurring between two genetic backgrounds: the starting enzyme (for example, wild type) and the intermediate variant on which the mutation occurred during the trajectory. We found that most trajectories exhibit positive epistasis, in which the mutational effect is more beneficial when it occurs later in the evolutionary trajectory. Approximately half (49%) of functional mutations were neutral or negative on the wild-type background, but became beneficial at a later stage in the trajectory, indicating that these functional mutations were not predictable from the initial starting point. While some cases of strong epistasis were associated with direct interaction between residues, many others were caused by long-range indirect interactions between mutations. Our work highlights the prevalence of epistasis in enzyme adaptive evolution, in particular positive epistasis, and suggests the necessity of incorporating mutational epistasis in protein engineering and design to create highly efficient catalysts.Keywords: epistasis; enzyme evolution; adaptive mutations; evolutionary constraints; directed evolution; protein engineering; rational design Abbreviations: DHFR, dihydrofolate reductase; MIC, minimum inhibitory concentration Additional Supporting Information may be found in the online version of this article.Significance Statement: Epistasis, that is, non-additive mutational effects, constrains the adaptive evolution of proteins, yet we lack a consensus view of its extent. To address this need, we performed a systematic survey of mutational epistasis occurring within nine adaptive enzyme trajectories. Of particular importance to protein engineering, our results quantify the unpredictability of functional mutations and emphasize the need to incorporate epistasis for the accurate prediction of mutational effects.

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Section: Resultsmentioning

confidence: 99%

How mutational epistasis impairs predictability in protein evolution and design

2016

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“…Specifically, for mutant SZ2155 (A85V/I86L/W110S) the respective single or double mutant generated by deconvolution showed S selectivity, but the triple mutant induced R selectivity. Such drastic mutational effects were observed in other studies upon using saturation mutagenesis at sites lining the enzyme binding pocket …”

Section: Resultsmentioning

confidence: 99%

Methodology Development in Directed Evolution: Exploring Options when Applying Triple‐Code Saturation Mutagenesis

Lonsdale

Yao

et al. 2018

ChemBioChem

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Directed evolution of stereo- or regioselective enzymes as catalysts in asymmetric transformations is of particular interest in organic synthesis. Upon evolving these biocatalysts, screening is the bottleneck. To beat the numbers problem most effectively, methods and strategies for building "small but smart" mutant libraries have been developed. Herein, we compared two different strategies regarding the application of triple-code saturation mutagenesis (TCSM) at multiresidue sites of the Thermoanaerobacter brockii alcohol dehydrogenase by using distinct reduced amino-acid alphabets. By using the synthetically difficult-to-reduce prochiral ketone tetrahydrofuran-3-one as a substrate, highly R- and S-selective variants were obtained (92-99 % ee) with minimal screening. The origin of stereoselectivity was provided by molecular dynamics analyses, which is discussed in terms of the Bürgi-Dunitz trajectory.

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“…This effect is notable for example in the design of peptide‐based organocatalysts, with a defined set of amino acids providing the highest enantioselectivities as compared to other epimeric sets of amino acids 2. Similarly, synergistic effects can be observed in enzymatic catalysis with a specific set of mutants being highly superior to others 3…”

Section: Influence Of the Ligand Configuration On The Enantioselectivmentioning

confidence: 96%

Synergistic Stereocontrol in the Enantioselective Ruthenium‐Catalyzed Sulfoxidation of Spirodithiolane‐Indolones

Zhong

Pöthig

Bach

2015

Chemistry A European J

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A chiral ruthenium catalyst was developed for the enantioselective sulfoxidation of the title compounds. The catalyst combines two elements of chirality, a chiral pybox ligand and a chiral bicylic lactam unit, to which the ligand is attached. The latter unit was shown to improve significantly the performance of the catalyst by exposing one of the two enantiotopic sulfur atoms to the active site via hydrogen-bond mediated coordination. Ten differently substituted substrates were converted into the respective sulfoxides in yields of 52-71 % and with ≥90 % ee.

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Extreme Synergistic Mutational Effects in the Directed Evolution of a Baeyer–Villiger Monooxygenase as Catalyst for Asymmetric Sulfoxidation

Cited by 70 publications

References 85 publications

How mutational epistasis impairs predictability in protein evolution and design

How mutational epistasis impairs predictability in protein evolution and design

Methodology Development in Directed Evolution: Exploring Options when Applying Triple‐Code Saturation Mutagenesis

Synergistic Stereocontrol in the Enantioselective Ruthenium‐Catalyzed Sulfoxidation of Spirodithiolane‐Indolones

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