Comparison of three enoate reductases and their potential use for biotransformations

Enoate reductases (ERs) selectively reduce carbon-carbon double bonds in a,p-unsaturated carbonyl compounds and thus can be employed to prepare enantiomerically pure aldehydes, ketones, and esters. Most known ERs, most notably Old Yellow Enzyme (OYE), are biochemically very well characterized. Some ERs have only been used in whole-cell systems, with endogenous ketoreductases often interfering with the ER activity. Not many ERs are biocatalytically characterized as to specificity and stability. Here, we cloned the genes and expressed three non-related ERs, two of them novel, in E. coli: XenA from Pseudomonas putida, KYEI from Kluyveromyces lactis, and Yers-ER from Yersinia bercovieri. All three proteins showed broad ER specificity and broad temperature and pH optima but different specificity patterns. All three proteins prefer NADPH as cofactor over NADH and are stable up to 40 degrees C. By coupling Yers-ER with glucose dehydrogenase (GDH) to recycle NADP(H), conversion of > 99% within one hour was obtained for the reduction of 2-cyclohexenone. Upon lowering the loadings of Yers-ER and GDH, we discovered rapid deactivation of either enzyme, especially of the thermostable GDH. We found that the presence of enone substrate, rather than oxygen or elevated temperature, is responsible for deactivation. In summary, we successfully demonstrate the wide specificity of enoate reductases for a range of alpha,beta-unsaturated carbonyl compounds as well as coupling to glucose dehydrogenase for recycling of NAD(P)(H); however, the stability limitations we found need to be overcome to envision large-scale use of ERs in synthesis.