Comparison of Three Enoate Reductases and their Potential Use for Biotransformations

Abstract

Enoate reductases (ERs) selectively reduce carbon-carbon double bonds in a,b-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, KYE1 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 8C. 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 a,b-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.

Carboxylation mechanism and stereochemistry of crotonyl-CoA carboxylase/reductase, a carboxylating enoyl-thioester reductase

“…Investigation of the carboxylation reaction of Ccr showed that CO 2 and not HCO 3 Ϫ is the carboxylating species. In principle, CO 2 can serve as electrophile in COC forming reactions on negatively polarized (or inverted) carbon atoms (5), whereas HCO 3 Ϫ is a nucleophile that has to be transformed into the carboxylating species by formation of a reactive carboxyphosphate upon ATP/phosphoester hydrolysis or as carboxyl-biotin to serve in enzymatic carboxylation reactions (44,45).…”

“…Enoate reductases are unique in their ability to reduce selectively CAC bonds in ␣,␤-unsaturated carbonyl compounds and to create thereby up to 2 stereogenic centers in the target molecule. This chemo-and stereoselectivity makes enoate reductases an important addition to the synthetic toolbox (1)(2)(3)(4).…”

An enoate reductase Achr-OYE4 from Achromobacter sp. JA81: characterization and application in asymmetric bioreduction of C=C bonds

“…Product formation could only be detected when the reaction was catalyzed with whole cells of recombinant E. coli expressing Achr-OYE4 (with E. coli cells harboring the empty vector as a control), suggesting possible deactivation of these substrates toward the purified enzyme. A previous report has indicated that the substrate plays a more important role in the deactivation of enoate reductase than oxygen or elevated temperature (Chaparro-Riggers et al 2007). In an attempt to eliminate the toxicity of the substrate, we tested biphasic systems that contain organic solvent with different partition coefficients.…”

“…Most characterized enoate reductases prefer NADPH over NADH as the cofactor (Chaparro-Riggers et al 2007;Toogood et al 2010), whereas only a few prefer NADH (Brige et al 2006;French and Bruce 1994), such as morphinone reductase, SYE1, and SYE3. In the present study, Achr-OYE4 displayed a preference for NADH on using all testing substrates, and the catalytic efficiency toward NADH Fig.…”

Activated α,β‐Unsaturated Aldehydes as Substrate of Dihydroxyacetone Phosphate (DHAP)‐Dependent Aldolases in the Context of a Multienzyme System

“…Interestingly, substrates 1-3a possess two activating groups (carbonyl groups) and they were also readily reduced by members of OYE family. [1] By contrast, other cyclohexanone derivatives 4-7a, which possess only one carbonyl group, had k cat /K m two orders of magnitude lower, despite being structurally similar to ketoisophorone 3a (Table 1, entries 4-8 vs. entry 3). In general, ketones 4-8a and 13a were relatively poor substrates under steady-state conditions (Table 1), whereas aldehydes 9-13a were reduced readily by PETNR.…”