Semi‐rational protein engineering of a novel ene‐reductase from <i>Galdieria sulphuraria</i> for asymmetric reduction of (<i>R</i>)‐carvone and ketoisophorone

Wu, Shijin; Wang, Bijiao; Yan, Hongde

doi:10.1002/bab.2391

Cited by 3 publications

(1 citation statement)

References 28 publications

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“…Mix I contained standard OYE substrates, (S)-carvone (1), ketoisophorone (2), cinnamaldehyde (3), and 4-phenylbut-3-yn-2-one (4), which served as controls for typical ene reductase activity. [30][31][32] Mix II consisted of various furanones (5-8) as precursors for high-value chiral synthons. 33 Mix III represented challenging substrates that known OYEs have little to no activity for.…”

Section: Selection Of Substrate Mixes Reaction Conditions and Charact...mentioning

confidence: 99%

The Hidden Biocatalytic Potential of the Old Yellow Enzyme Family

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Iamurri

Keshavarz-Joud

et al. 2023

Preprint

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The rapid advancement of sequencing technology has created an immense reservoir of protein sequence-function information that has yet to be fully utilized for fundamental or biocatalytic applications. For example, ene reductases from the "old yellow enzyme" (OYE) family catalyze the asymmetric hydrogenation of activated alkenes with enhanced stereoselectivity - key transformations for sustainable production of pharmaceutical and industrial synthons. Despite the proven biocatalytic application, the OYE family remains relatively underexplored with only 0.1% of identified members having any experimental characterization. Here, a platform of integrated bioinformatics and synthetic biology techniques was employed to systematically organize and screen the natural diversity of the OYE family. Using protein similarity networks, the known and unknown regions of the >115,000 members of the OYE family were broadly explored to identify phylogenetic and sequence-based trends. From this analysis, 118 novel enzymes were characterized across the family to broadly explore and expand the biocatalytic performance and substrate scope of known OYEs. Over a dozen novel enzymes were identified exhibiting enhanced catalytic activity or altered stereospecificity. Beyond well-established ene reduction, we detected widespread occurrence of oxidative chemistry amongst OYE family members at ambient conditions. Crystallography studies of selected OYEs yielded structures for two enzymes, contributing to a better understanding of their unique performance. Their structures revealed an unusual loop conformation within a novel OYE subclass. Overall, our study significantly expands the known functional and chemical diversity of OYEs while identifying superior biocatalysts for asymmetric reduction and oxidation.

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Section: Selection Of Substrate Mixes Reaction Conditions and Charact...mentioning

confidence: 99%

The Hidden Biocatalytic Potential of the Old Yellow Enzyme Family

White

Iamurri

Keshavarz-Joud

et al. 2023

Preprint

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Old Yellow Enzymes as Oxime Reductases: New Variants by Substrate‐Based Enzyme Engineering

Polidori,

Breukelaar,

Stelzer

et al. 2024

ChemCatChem

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The reduction of oximes was recently identified as a promiscuous activity of Old Yellow Enzymes (OYEs). This reaction involves a two‐step reduction of α‐oxime‐ß‐ketoesters to the corresponding amines, which spontaneously dimerise to yield pyrazine derivatives. This biotransformation is currently limited to substrates with small substituents like methyl/ethyl on the keto moiety. We used a structure‐based approach to engineer 12‐oxophytodienoate reductase 3 (OPR3) from Solanum lycopersicum as a prototypical OYE to accept oximes with bulkier substituents. To this end, three single and two double variants were prepared and tested on six oxime substrates. The engineered variants indeed showed activity on some of the bulkier substrates, which had not been converted at all by the wild‐type enzyme, including the diester compound diethyl‐2‐(hydroximino)malonate. While we were unable to identify variants capable of converting substrates with branched and aromatic substituents, the results demonstrate the validity of our engineering approach suggesting potential pathways for expanding the substrate scope of OYEs.

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