Catalytic Performance of a Class III Old Yellow Enzyme and Its Cysteine Variants.

Anika Scholtissek,Adrie H Westphal,Willem J H Van Berkel,Dirk Tischler,Caroline E Paul,Eric Gädke

doi:10.3389/fmicb.2018.02410

Abstract

Class III old yellow enzymes (OYEs) contain a conserved cysteine in their active sites. To address the role of this cysteine in OYE-mediated asymmetric synthesis, we have studied the biocatalytic properties of OYERo2a from Rhodococcus opacus 1CP (WT) as well as its engineered variants C25A, C25S and C25G. OYERo2a in its redox resting state (oxidized form) is irreversibly inactivated by N-methylmaleimide. As anticipated, inactivation does not occur with the Cys variants. Steady-state kinetics with this maleimide substrate revealed that C25S and C25G doubled the turnover frequency (kcat) while showing increased KM values compared to WT, and that C25A performed more similar to WT. Applying the substrate 2-cyclohexen-1-one, the Cys variants were less active and less efficient than WT. OYERo2a and its Cys variants showed different activities with NADPH, the natural reductant. The variants did bind NADPH less well but kcat was significantly increased. The most efficient variant was C25G. Replacement of NADPH with the cost-effective synthetic cofactor 1-benzyl-1,4-dihydronicotinamide (BNAH) drastically changed the catalytic behavior. Again C25G was most active and showed a similar efficiency as WT. Biocatalysis experiments showed that OYERo2a, C25S, and C25G converted N-phenyl-2-methylmaleimide equally well (81–84%) with an enantiomeric excess (ee) of more than 99% for the R-product. With cyclic ketones, the highest conversion (89%) and ee (>99%) was observed for the reaction of WT with R-carvone. A remarkable poor conversion of cyclic ketones occurred with C25G. In summary, we established that the generation of a cysteine-free enzyme and cofactor optimization allows the development of more robust class III OYEs.

Highlights

Protein engineering is a powerful tool to improve the catalytic properties of enzymes
The mutations introduced at Cys25 did not change the model and the active site construction, which is in accordance with results obtained for xenobiotic reductase A (XenA), where no structural perturbations were observed upon changing Cys25 (Spiegelhauer et al, 2010)
The covalent modification reaction with this maleimide was completely prevented when Ser/Ala/Gly protein variants were applied. Because this property is of benefit for biocatalytic applications, we addressed the catalytic performance of the OYERo2 generated a protein variant (OYERo2a) Cys variants

Summary

Introduction

Protein engineering is a powerful tool to improve the catalytic properties of enzymes. The FMN-containing OYEs catalyze the selective reduction of activated α,β-unsaturated alkenes yielding valuable alkanes containing one or two chiral carbon centers (Scheme 1). Capable of catalyzing the asymmetric trans-hydrogenation of various alkene substrates such as cyclic enones, maleimides, aldehydes, or (di)-carboxylic acids (Swiderska and Stewart, 2006a; Nivinskas et al, 2008; Toogood et al, 2008; Fryszkowska et al, 2009; Gao et al, 2012; Fu et al, 2013; Riedel et al, 2015; Scholtissek et al, 2017b; Toogoood and Scrutton, 2018), ERs promise potential applications in industrial processes due to their exquisite regio-, stereo- and enantioselectivity. The FMN cofactor receives electrons from NAD(P)H, but ERs accept nicotinamide coenzyme biomimetics (NCBs) (Knaus et al, 2016; Scholtissek et al, 2017a)

Results

Discussion

Conclusion