Asymmetric Bioreduction of CC Bonds using Enoate Reductases OPR1, OPR3 and YqjM: Enzyme‐Based Stereocontrol

Abstract

Three cloned enoate reductases from the "old yellow enzyme" family of flavoproteins were investigated in the asymmetric bioreduction of activated alkenes. 12-Oxophytodienoate reductase isoenzymes OPR1 and OPR3 from Lycopersicon esculentum (tomato), and YqjM from Bacillus subtilis displayed a remarkably broad substrate spectrum by reducing a,b-unsaturated aldehydes, ketones, maleimides and nitroalkenes. The reaction proceeded with absolute chemoselectivity -only the conjugated C=C bond was reduced, while isolated olefins and carbonyl groups remained intact -with excellent stereoselectivities (ees up to > 99%). Upon reduction of a nitroalkene, the stereochemical outcome could be determined via choice of the appropriate enzyme (OPR1 versus OPR3 or YqjM), which furnished the corresponding enantiomeric nitroalkanes in excellent ee. Molecular modelling suggests that this "enzymebased stereocontrol" is caused by subtle differences within the active site geometries.

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

“…A similar time-dependent loss of enantiopurity for these compounds was seen with other OYEs, although it was not investigated in detail. [2,3] Since the enzymatic reaction with these substrates was complete after 24 h, we concluded that the loss of optical purity was due to racemisation of products with a stereogenic centre at Cα. The apparent rate of racemisation varied depending on the nature of the product.…”

“…This unexpected change of enantioselectivity for formation of 2-methylcyclopentanone 17b has also been reported for other OYEs, although no explanation for this observation was given. [2][3][4] In light of recent work by Stewart et al [5] it is possible that substrate 17a may bind in a 'flipped' orientation, while maintaining an optimal Cβ-FMN N5 distance and angle (105°), [21] similar to that seen for OYE1 mutant W116I. [5] PETNR proved to be highly efficient and highly stereoselective in the reduction of 2-methylmaleimide derivatives 1a-b giving the respective (2R)-products in quantitative yields and high enantioselectivities (>99% ee, Table 2 and Scheme 1c).…”

“…[2][3][4] As previous studies have shown that the nature of the cofactor or cofactor regeneration system affects the enantiopurity and even the absolute configuration of the product, [2][3][4] we studied the effect of using NADP + /G6PDH as cofactor recycling system vs. reactions with excess NADPH (Table 3). Both reaction conditions proved to be equally efficient (inferred from similar yields and conversion) and the products had similar optical purities after 24 h. When we extended the reaction time for the NADP + /G6PDH system (48-72 h), we observed high optical purity of the reduction products possessing a stereogenic centre at Cβ (>99% ee for…”

Identification of enzymes responsible for the reduction of geraniol to citronellol

“…Successful attempts in the chemo-and stereoselective reduction of activated alkenes were reported by using cloned yeast OYE1 from Saccharomyces carlsbergensis, [5][6][7] , OYE2 and OYE3 from S. cerevisiae, [6][7][8] CmOYE from Candida macedoniensis; 9 and bacterial OYE homologues YqjM from Bacillus subtilis, NCR from Zymomonas mobilis; 6,10 and plant OYE homologues LeOPR1 and LeOPR3 from Lycopersicon esculentum (tomato). 10,11 The plant 12-oxophytodienoate reductase (OPR) is involved in the reduction of (9S,13S)-12-oxophytodienoate to the corresponding cyclopentanoic acid OPC-8:0 for jasmonate biosynthesis. 12 By using the cloned OPR1 and OPR3 from tomato, a striking change of stereoselectivity and a remarkably broad substrate spectrum were achieved for asymmetric reduction.…”

“…12 By using the cloned OPR1 and OPR3 from tomato, a striking change of stereoselectivity and a remarkably broad substrate spectrum were achieved for asymmetric reduction. 10,11 Although the above OYE enzymes have been biochemically well characterized, they have never been investigated for the capacity of reducing the allylic alcohol double bond in geraniol, because alcohols are not typically favored as activating groups for OYEs. 3 In our effort to broaden the applicability of plant OPR, we came across its catalytic activity of converting geraniol to citronellol as a sole product (Scheme).…”