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

Abstract

Chemo-and stereoselective reductions are important reactions in chemistry and biology, and reductases from biological sources are increasingly applied in organic synthesis. In contrast, carboxylases are used only sporadically. We recently described crotonyl-CoA carboxylase/reductase, which catalyzes the reduction of (E)-crotonyl-CoA to butyryl-CoA but also the reductive carboxylation of (E)-crotonyl-CoA to ethylmalonyl-CoA. In this study, the complete stereochemical course of both reactions was investigated in detail. The pro-(4R) hydrogen of NADPH is transferred in both reactions to the re face of the C3 position of crotonyl-CoA. In the course of the carboxylation reaction, carbon dioxide is incorporated in anti fashion at the C2 atom of crotonyl-CoA. For the reduction reaction that yields butyryl-CoA, a solvent proton is added in anti fashion instead of the CO2. Amino acid sequence analysis showed that crotonyl-CoA carboxylase/reductase is a member of the medium-chain dehydrogenase/reductase superfamily and shares the same phylogenetic origin. The stereospecificity of the hydride transfer from NAD(P)H within this superfamily is highly conserved, although the substrates and reduction reactions catalyzed by its individual representatives differ quite considerably. Our findings led to a reassessment of the stereospecificity of enoyl(-thioester) reductases and related enzymes with respect to their amino acid sequence, revealing a general pattern of stereospecificity that allows the prediction of the stereochemistry of the hydride transfer for enoyl reductases of unknown specificity. Further considerations on the reaction mechanism indicated that crotonyl-CoA carboxylase/reductase may have evolved from enoyl-CoA reductases. This may be useful for protein engineering of enoyl reductases and their application in biocatalysis.alcohol dehydrogenase ͉ biocatalysis ͉ enoyl reductase

A warm welcome for alternative CO2 fixation pathways in microbial biotechnology

“…In addition, these MOG pathways involve only oxygen‐tolerant enzymes and have predicted lower ATP costs than the Calvin cycle. In addition, synthetic pathways based on crotonyl‐CoA carboxylases, the fastest known carboxylases, may be highly promising to apply in synthetic CO 2 fixation pathways (Erb et al ., 2009). …”

Global and Local Reactivity Descriptors Based on Quadratic and Linear Energy Models for α,β-Unsaturated Organic Compounds

“…Recently, it has been shown that they also participate in the fixation of carbon dioxide in alternative carbon fixation cycles [1,23,24,25]. In this cycle one of the important step is the reduction of the α, β -unsaturated thioester by the reduced form of nicotinamide adenosine dinucleotide phosphate (NADPH) to produce an enolate that binds CO 2 (see Figure 1).…”

“…In this cycle one of the important step is the reduction of the α, β -unsaturated thioester by the reduced form of nicotinamide adenosine dinucleotide phosphate (NADPH) to produce an enolate that binds CO 2 (see Figure 1). This reaction is catalyzed by Crotonyl -CoA -carboxylase yielding enantiomeric products and has been widely studied by Erb et al [1]. Recently, enzymes of the same family were shown to also catalyze various thioesters with different substitutions of the alkyl chain [26].…”

“…The ability of these descriptors to predict the reactivity of molecules with more than one reactive site was first studied on a set of α, β -unsaturated ketones, where experimental rate constants for the nucleophilic attack is known. With the validated descriptors the reactivity of α, β -unsaturated carboxylic compounds with different heteroatoms as α, β -unsaturated thioesters, esters and amides as alternative substrates for the enzymatic CO 2 fixation studied experimentally by Erb et al [1] was addressed. The carbon dioxide fixation involves the reduction of the neutral α, β -unsaturated carboxylic compounds by a nucleophilic attack of a hydride anion from NADPH and the following electrophilic attack by carbon dioxide.…”

“…The carbon dioxide fixation involves the reduction of the neutral α, β -unsaturated carboxylic compounds by a nucleophilic attack of a hydride anion from NADPH and the following electrophilic attack by carbon dioxide. It was 1 found that condensed values of the linear fukui function within the fragment of molecular response approximation describe best the reactivity of α, β -unsaturated ketones.…”