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

Abstract

We show that pentaerythritol tetranitrate reductase (PETNR), a member of the 'ene' reductase old yellow enzyme family, catalyses the asymmetric reduction of a variety of industrially relevant activated α,β-unsaturated alkenes including enones, enals, maleimides and nitroalkenes. We have rationalised the broad substrate specificity and stereochemical outcome of these reductions by reference to molecular models of enzyme-substrate complexes based on the crystal complex of the PETNR with 2-cyclohexenone 4a. The optical purity of products is variable (49-99% ee), depending on the substrate type and nature of substituents. Generally, high enantioselectivity was observed for reaction products with stereogenic centres at Cβ (>99% ee). However, for the substrates existing in two isomeric forms (e.g., citral 11a or nitroalkenes 18-19a), an enantiodivergent course of the reduction of E/Z-forms may lead to lower enantiopurities of the products. We also demonstrate that the poor optical purity obtained for products with stereogenic centres at Cα is due to non-enzymatic racemisation. In reactions with ketoisophorone 3a we show that product racemisation is prevented through reaction optimisation, specifically by shortening reaction time and through control of solution pH. We suggest this as a general strategy for improved recovery of optically pure products with other biocatalytic conversions where there is potential for product racemisation.

Design of Artificial Metabolisms in Layered Nanomaterials for the Enzymatic Synthesis of Phosphorylated Sugars

“…Given that DHAP is a key substrate for DHAP‐dependent aldolases to prepare various 1‐phosphorylated sugars, the efficiency of the PK Gstea –DHAK@Mg 2 Al biohybrid was evaluated for four reactions by using commercially available rabbit muscle aldolase (RAMA, 3 S ,4 R stereoselective) and rhamnulose aldolase (RhuA, 3 R ,4 S stereoselective) produced and purified as already described . These aldolases were used with two acceptors, glycolaldehyde and d ‐glyceraldehyde (Scheme , see also Scheme S3).…”

Multi‐Enzymatic Cascade Reactions: Overview and Perspectives

“…Acetate kinase served for ATP regeneration. The in-situ DHAP generation by this approach can be utilized for the synthesis of unusual sugars with a variety of different aldehydes; [97] however, the authors report that ethyl 3-methyl-4-oxocrotonate inhibited DHA-kinase activity and therefore re- that possesses both aldolase and kinase activities. [98,99] The fusion protein consists of a monomeric fructose-1,6-diphosphate aldolase from Staphylococcus carnosus and a homodimeric dihydroxyacetone kinase from C. freundii linked by five amino acids.…”

Current Trends in Asymmetric Synthesis with Aldolases

“…[20] For this reason, numerous methods were developed for the enzymatic in situ preparation of 42, including cascade enzymatic reactions. [84] Most recently, a bifunctional fusion protein between the dihydroxyacetone kinase (DHAK) from Citrobacter freundii and FruA from Staphylococcus carnosus has been reported which catalyzes the phosphorylation of 43 and its subsequent aldol addition with 20-fold improved kinetics as compared to the use of separate enzymes (Scheme 15). [84] Most recently, a bifunctional fusion protein between the dihydroxyacetone kinase (DHAK) from Citrobacter freundii and FruA from Staphylococcus carnosus has been reported which catalyzes the phosphorylation of 43 and its subsequent aldol addition with 20-fold improved kinetics as compared to the use of separate enzymes (Scheme 15).…”

“…[83] Multienzyme systems using a dihydroxyacetone kinase (DHAK) from Citrobacter freundii, and FruA from rabbit muscle (RAMA) or l-rhamnulose-1-phosphate aldolase (RhuA) or FucA from E. coli and acetate kinase (AK) for adenosine triphosphate (ATP) regeneration, have been studied and proved to be efficient for aldol addition reactions of DHAP to several aldehydes. [84] Most recently, a bifunctional fusion protein between the dihydroxyacetone kinase (DHAK) from Citrobacter freundii and FruA from Staphylococcus carnosus has been reported which catalyzes the phosphorylation of 43 and its subsequent aldol addition with 20-fold improved kinetics as compared to the use of separate enzymes (Scheme 15). [85,86] A novel method was reported for the generation of DHAP from DHA based on the use of the acid phosphatase from Shigella flexneri (PhoN-Sf) using pyrophosphate (PPi) as phosphate donor.…”