Shifting the Substrate Scope of an Ene/Yne‐Reductase by Loop Engineering DOI Creative Commons

Dominik Karrer,

Erika Wedler,

Carolin Mewe

et al.

ChemCatChem, Journal Year: 2025, Volume and Issue: unknown

Published: June 5, 2025

Abstract Medium‐chain dehydrogenase/reductase‐superfamily related (MDR) ene‐reductases are highly neglected biocatalysts that have not yet been systematically studied in terms of rational protein engineering. Therefore, we crystallized the MDR‐related CaeEnR1 its apo‐ and binary‐complex searched for secondary structures might influence enzyme activity toward specific substrate classes. By comparing binary‐complexes, a flexible active‐site loop was identified is involved shaping product cleft as well pocket. Furthermore, could show an undergoes drastic re‐arrangement after cofactor/substrate binding, revealing open–closed mechanism. Based on these results, subjected this to alanine‐scan. The hits were then designed substituted cinnamaldehyde‐like substrates aliphatic/aromatic alkyne. From all tested amino acids P64, Y69, S70 most influential acid residues. Particularly double mutations P64F/Y69A Y69F/S70A showed up 20‐fold increase conversion rates substrates. In contrast, 2‐fold aliphatic aromatic alkyne achieved with S70F variant. With study state foundation engineering ERs which opens new pathway tailored biocatalysts.

Language: Английский

Shifting the Substrate Scope of an Ene/Yne‐Reductase by Loop Engineering DOI Creative Commons

Dominik Karrer,

Erika Wedler,

Carolin Mewe

et al.

ChemCatChem, Journal Year: 2025, Volume and Issue: unknown

Published: June 5, 2025

Abstract Medium‐chain dehydrogenase/reductase‐superfamily related (MDR) ene‐reductases are highly neglected biocatalysts that have not yet been systematically studied in terms of rational protein engineering. Therefore, we crystallized the MDR‐related CaeEnR1 its apo‐ and binary‐complex searched for secondary structures might influence enzyme activity toward specific substrate classes. By comparing binary‐complexes, a flexible active‐site loop was identified is involved shaping product cleft as well pocket. Furthermore, could show an undergoes drastic re‐arrangement after cofactor/substrate binding, revealing open–closed mechanism. Based on these results, subjected this to alanine‐scan. The hits were then designed substituted cinnamaldehyde‐like substrates aliphatic/aromatic alkyne. From all tested amino acids P64, Y69, S70 most influential acid residues. Particularly double mutations P64F/Y69A Y69F/S70A showed up 20‐fold increase conversion rates substrates. In contrast, 2‐fold aliphatic aromatic alkyne achieved with S70F variant. With study state foundation engineering ERs which opens new pathway tailored biocatalysts.

Language: Английский

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