Functional characterization and stability improvement of a ‘thermophilic-like’ ene-reductase from Rhodococcus opacus 1CP

Abstract

Ene-reductases (ERs) are widely applied for the asymmetric synthesis of relevant industrial chemicals. A novel ER OYERo2 was found within a set of 14 putative old yellow enzymes (OYEs) obtained by genome mining of the actinobacterium Rhodococcus opacus 1CP. Multiple sequence alignment suggested that the enzyme belongs to the group of ‘thermophilic-like’ OYEs. OYERo2 was produced in Escherichia coli and biochemically characterized. The enzyme is strongly NADPH dependent and uses non-covalently bound FMNH2 for the reduction of activated α,β-unsaturated alkenes. In the active form OYERo2 is a dimer. Optimal catalysis occurs at pH 7.3 and 37°C. OYERo2 showed highest specific activities (45-50 U mg-1) on maleimides, which are efficiently converted to the corresponding succinimides. The OYERo2-mediated reduction of prochiral alkenes afforded the (R)-products with excellent optical purity (ee > 99%). OYERo2 is not as thermo-resistant as related OYEs. Introduction of a characteristic intermolecular salt bridge by site-specific mutagenesis raised the half-life of enzyme inactivation at 32°C from 28 to 87 min and improved the tolerance toward organic co-solvents. The suitability of OYERo2 for application in industrial biocatalysis is discussed.

Old Yellow Enzyme-Catalysed Asymmetric Hydrogenation: Linking Family Roots with Improved Catalysis

“…Even higher species as octamers and dodecamers, emerged from functional homodimers, were observed for TOYE and TsOYE [77,79]. A shift between higher multiple oligomeric states was noticed depending on the protein concentration [22,77]. On the other hand, members of class I and class II were found to exclusively occur as monomers or homodimers [14,83,109].…”

“…A remarkable property observed for class III OYEs is their occurrence in solution as homodimers and homotetramers [22,76,105]. Even higher species as octamers and dodecamers, emerged from functional homodimers, were observed for TOYE and TsOYE [77,79].…”

“…Nevertheless, the enzymes AtOPR1-AtOPR3 from Arabidopsis thaliana [65,66], and LeOPR1-LeOPR3 from Solanum lycopersicum (tomato) [67,68], were characterised according to their structure, function and physiological role. Two thirds of the characterised OYEs have been obtained from various classes of bacteria including proteobacteria (28%) [69,70], actinobacteria (5%) [22,71,72], bacteroidetes (5%) [73,74], firmicutes (10%) [75][76][77], deinococcus-thermus (3%) [78][79][80], and cyanobacteria (17%) [21,81].…”

“…Catalysts 2017, 7, 130 2 of 24 activated alkenes with an electron withdrawing group (EWG) such as aldehyde, ketone, anhydride [8,10,11], nitro [9,12,13], (di)ester [8,[14][15][16][17], (di)carboxylic acid [18][19][20], cyclic imide [21][22][23], nitrile [24], β-cyanoacrylate [25], β-nitroacrylate [26], and several other functional groups [27]. There are many examples of the high industrial potential of OYEs for the synthesis of valuable target products [28][29][30][31].…”

“…In a first study, YqjM, TsOYE and RmOYE were screened against NCBs 1-5 ( Figure 1) [52], followed by more extensive kinetic studies with a panel of OYEs [59]. Depending on the applied NCBs, OYEs differ in their reaction rates and the catalytic efficiency (kcat/Km) is at times even higher with NCBs than with the natural coenzyme, as discussed further in Section 4 [22,58,59].…”