ORCID Profile
0000-0002-5678-6394
Current Organisation
Korea Institute of Science and Technology
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Publisher: Wiley
Date: 22-03-2023
Publisher: Wiley
Date: 08-03-2023
Abstract: Asymmetric reduction by ene‐reductases has received considerable attention in recent decades. While several enzyme families possess ene‐reductase activity, the Old Yellow Enzyme (OYE) family has received the most scientific and industrial attention. However, there is a limited substrate range and few stereocomplementary pairs of current ene‐reductases, necessitating the development of a complementary class. Flavin/deazaflavin oxidoreductases (FDORs) that use the uncommon cofactor F 420 have recently gained attention as ene‐reductases for use in biocatalysis due to their stereocomplementarity with OYEs. Although the enzymes of the FDOR‐As sub‐group have been characterized in this context and reported to catalyse ene‐reductions enantioselectively, enzymes from the similarly large, but more erse, FDOR‐B sub‐group have not been investigated in this context. In this study, we investigated the activity of eight FDOR‐B enzymes distributed across this sub‐group, evaluating their specific activity, kinetic properties, and stereoselectivity against α,β‐unsaturated compounds. The stereochemical outcomes of the FDOR‐Bs are compared with enzymes of the FDOR‐A sub‐group and OYE family. Computational modelling and induced‐fit docking are used to rationalize the observed catalytic behaviour and proposed a catalytic mechanism.
Publisher: Cold Spring Harbor Laboratory
Date: 21-02-2023
DOI: 10.1101/2023.02.21.529347
Abstract: Ene-reductases from the Flavin/Deazaflavin Oxidoreductase (FDOR) family have potential value in biocatalysis as they typically exhibit complementary stereoselectivity to the widely utilized Old Yellow Enzyme (OYE) family, yet they are comparatively poorly understood at a mechanistic level. Here, we use a rational design approach to generate a library of 46 active site mutants of two FDORs from Mycobacterium smegmatis and examine the effects on conversion and stereoselectivity against a panel of substrates. Analysis of the effects of these mutations on stereoselectivity across all substrates revealed that the catalytic mechanism is highly sensitive to the polarity of the immediate active site. A conserved active site tyrosine in these enzymes, which does not serve as the proton donor, strongly affects stereochemical outcomes with Cα- (but not Cβ-) substituted substrates. Notably, a Tyr-Met mutation at this position reversed the diastereomeric excess ( de ) with ( R )-carvone from 85.3% to −17.3% (cis/trans). Additionally, this mutation significantly increases activity with (1 S )- verbenone. Finally, we show that the altered stereoselectivity is not due to a “flipped” substrate binding mode in these mutants, but rather that the hydrogenation mode is altered to favor syn relative to anti addition. These results show that the FDORs are highly engineerable and that, despite their superficial similarity, the OYE and FDOR families differ in crucial mechanistic aspects.
Publisher: Cold Spring Harbor Laboratory
Date: 05-11-2022
DOI: 10.1101/2022.11.05.515281
Abstract: The stereoselective reduction of alkenes conjugated to electron-withdrawing groups by ene-reductases has been extensively applied to the commercial preparation of fine chemicals. Although several different enzyme families are known to possess ene-reductase activity, the Old Yellow Enzyme (OYE) family has been the most thoroughly investigated. Recently, it was shown that a subset of ene-reductases belonging to the flavin/deazaflavin oxidoreductase (FDOR) superfamily exhibit enantioselectivity that is generally complementary to that seen in the OYE family. These enzymes belong to one of several FDOR subgroups that use the unusual deazaflavin cofactor F 420 . Here, we explore several enzymes of the FDOR-A subgroup, characterizing their substrate range and enantioselectivity, including the complete conversion of both isomers of citral to (R )-citronellel with 99% ee . Protein crystallography combined with computational docking has allowed the observed stereoselectivity to be mechanistically rationalized for two enzymes. These findings add further support for the FDOR and OYE families of ene-reductases being generally stereocomplementary to each other and highlight their potential value in asymmetric ene-reduction.
Location: Korea, Republic of
No related grants have been discovered for Suk Woo Kang.