Asymmetric Ene‐Reduction by F₄₂₀‐Dependent Oxidoreductases B (FDOR‐B) from Mycobacterium smegmatis

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 F420 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 diverse, 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.

Chemistry of Coenzyme F420 in Environment

“…This is revolutionizing the diversity and metabolic potential of the archaea in a wide variety of environments, an important step toward understanding their ecological role and industrial applications. [12][13][14] The ancient coenzyme F420 is one of the important members of the ancient coenzymes and cofactors that participate in methanogenesis, sulfate-reduction, and methanotrophic reactions in archaea. 15,16 The coenzyme F420 is also present in a wide range of actinomycetes, mycobacteria, and other bacteria.…”

Active site mutations of F420-dependent alkene reductases reverse stereoselectivity

“…[6][7][8] More recently, ene-reductases that utilize the deazaflavin cofactor F420 have been shown to reduce their substrates in a regio-and enantioselective manner that is often stereocomplementary to OYEs. [9][10][11] These enzymes belong to the flavin/deazaflavin oxidoreductase (FDOR) family of split β-barrel proteins. 12 While the cofactor F420 occurs only in a limited number of prokaryotes, elucidation of its biosynthesis and metabolic engineering has enabled its production in the industrial workhorse Escherichia coli (in which it is not natively produced) at high titers, creating opportunities for its use as a bio-orthogonal cofactor in biocatalysis.…”

“…24 We recently reported detailed characterization of the ene-reductase activity and substrate range of the FDOR enzymes from the -A and -B subgroups. 10, 11 Among the enzymes we have characterized, the FDOR-A enzymes MSMEG_2027 and MSMEG_2850 from Mycobacterium smegmatis demonstrated the best combination of activity and broad substrate range. 10 Using X-ray crystallography and computational substrate docking it was possible to identify the preferred binding mode of substrates in the active site, which when combined with the experimentally observed products suggested that reduction proceeds with net anti addition of H 2 across the double bond, as is most commonly observed in the OYE family.…”