Asymmetric Ene-Reduction of α,β-Unsaturated Compounds by F₄₂₀-Dependent Oxidoreductases A Enzymes from Mycobacterium smegmatis

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 with 20 different compounds, identifying enzymes (MSMEG_2027 and MSMEG_2850) that could reduce a wide range of compounds stereoselectively. For example, MSMEG_2027 catalyzed the complete conversion of both isomers of citral to (R)-citronellal with 99% ee, while MSMEG_2850 catalyzed complete conversion of ketoisophorone to (S)-levodione 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 displaying general stereocomplementarity to each other and highlight their potential value in asymmetric ene-reduction.

Active site mutations of F420-dependent alkene reductases reverse stereoselectivity

“…17 The substrates that were turned over appreciably by MSMEG_2027-S67A were those for which the wildtype enzyme has relatively high specific activity. 10 This was also the case with the M54Q variant and the N11W/Y, F29R, F64N and F121Y variants of MSMEG_2850. Interestingly, the V30R mutation in MSMEG_2027 was not as deleterious as the equivalent mutation in MSMEG_2850 (F29R).…”

“…We previously reported the crystal structures of MSMEG_2027 bound with F420 (PDB 6WTA) and MSMEG_2850 in the apo state (PBD 8D4W) from which we generated holoenzyme models and performed computational substrate docking. 10 For our rational protein engineering approach, we selected (R)-(−)-carvone (1a) as a model substrate because (i) the bulky 5-substituent should strongly direct binding orientation;…”

“…This compound has two carbonyl groups that could bind in the oxyanion hole, and we previously obtained docking poses of both orientations with the wildtype enzymes. 10 With the C4-carbonyl as the activating group protonation occurs at unsubstituted C3, whereas with the C1-carbonyl as the activating group protonation take place at methylsubstituted C2. Through net anti addition of H2, both poses would yield the observed major product (S)-7b.…”

“…[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.…”

“…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 H2 across the double bond, as is most commonly observed in the OYE family. 3,[25][26][27][28] Although mutagenesis has been used to interrogate FDORs involved in specific physiologically relevant activities such as reduction of biliverdin and antitubercular nitroimidazole prodrugs, 18,20 there are few reports of engineering FDORs as biocatalysts for the reduction of alkenes or using mutagenesis to probe their structurefunction relationships in a broad manner.…”