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 CO(2). 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.

Fig. 1.

Crotonyl-CoA carboxylase/reductase. Reactions catalyzed by Ccr. Properties of crotonyl-CoA carboxylase/reductase are shown in Table 1.

Fig. 2.

Determination of the carboxylating species of the Ccr reaction. The oxidation of NADPH during the reductive carboxylation of crotonyl-CoA to ethylmalonyl-CoA was followed spectrophotometrically at 360 nm. To determine the carboxylating species, the reaction was started either with dissolved CO₂ (black solid line), or HCO₃⁻ (gray solid line) at 15 °C, a temperature, at which the hydration of CO₂/dehydration of HCO₃⁻ is slow. As control, the reaction was started also with dissolved CO₂ (black dotted line) or HCO₃⁻ (gray dotted line) in the presence of carbonic anhydrase (C.A.), an enzyme that catalyzes the reversible hydration of CO₂.

Fig. 3.

Determination of the stereochemistry of the carboxylation product. (A) Acryloyl-CoA was incubated with NADPH, Ccr, and ¹⁴C-labeled NaHCO₃, resulting in radioactive-labeled methylmalonyl-CoA as shown by HPLC and radioactive monitoring. (B–D) To determine the stereochemistry of the product, this methylmalonyl-CoA was subsequently incubated for 1 min with methylmalonyl-CoA epimerase (Epi) (B), (2R)-methylmalonyl-CoA mutase (Mcm) (C), or a combination of both enzymes (D). The formation of radioactive labeled products was followed by HPLC.

Fig. 4.

Determination of the stereochemistry of the hydride transfer (carboxylase reaction). The stereospecificity of the hydride transfer from the prochiral C4 position of the nicotinamide was determined by using stereospecifically labeled NADPH. Crotonyl-CoA was incubated in the presence of HCO₃⁻/CO₂ with unlabeled NADPH (A), [²H]-(4S)-NADPH (B), or [²H]-(4R)-NADPH (C). The products were analyzed by HPLC-MS. The corresponding mass spectra of the ethylmalonyl-CoA species formed are shown in detail.

Fig. 5.

Determination of the stereochemistry of the reductase reaction. (A and B) Stereochemistry at C3. Crotonyl-CoA was incubated in the absence of HCO₃^−]/CO₂ with Ccr and either NADPH or [²H]-(4R)-NADPH, and the butyryl-CoA species formed were analyzed by HPLC-MS. The corresponding HPLC chromatograms (A1, B1) are shown together with the detailed mass spectra of the respective butyryl-CoA peaks. The butyryl-CoA species were isolated by preparative HPLC from the reaction mixture and converted back to crotonyl-CoA by pro-(2R), pro-(3R)-specific butyryl-CoA dehydrogenase (BDH) from pig liver mitochondria, to determine the absolute stereochemistry of the label incorporated. The corresponding HPLC chromatograms (A2, B2) and the respective mass spectra of crotonyl-CoA species are shown. (C and D) Stereochemistry at C2. Crotonyl-CoA was incubated in the absence of HCO₃⁻/CO₂ in [²H]₂O with Ccr and either unlabeled NADPH or [²H]-(4R)-NADPH and the butyryl-CoA species formed were analyzed by HPLC-MS. The corresponding HPLC chromatograms (C1, D1) and detailed mass spectra of the butyryl-CoA peaks are shown. After isolation of the butyryl-CoA species from the reaction mixtures by preparative HPLC, the CoA-esters were converted back to crotonyl-CoA by butyryl-CoA dehydrogenase (BDH) to determine the absolute stereochemistry of the label incorporated. The HPLC chromatograms (C2, D2) and mass spectra of crotonyl-CoA species are shown in detail.