Fifty years ago, Kornberg and Krebs established the glyoxylate cycle as the pathway for the synthesis of cell constituents from C2-units. However, since then, many bacteria have been described that do not contain isocitrate lyase, the key enzyme of this pathway. Here, a pathway termed the ethylmalonyl-CoA pathway operating in such organisms is described. Isotopically labeled acetate and bicarbonate were transformed to ethylmalonyl-CoA by cell extracts of acetate-grown, isocitrate lyase-negative Rhodobacter sphaeroides as determined by NMR spectroscopy. Crotonyl-CoA carboxylase/reductase, catalyzing crotonyl-CoA ؉ CO 2 ؉ NADPH 3 ethylmalonyl-CoA ؊ ؉ NADP ؉ was identified as the key enzyme of the ethylmalonyl-CoA pathway. The reductive carboxylation of an enoyl-thioester is a unique biochemical reaction, unprecedented in biology. The enzyme from R. sphaeroides was heterologously produced in Escherichia coli and characterized. Crotonyl-CoA carboxylase/reductase (or its gene) can be used as a marker for the presence of the ethylmalonyl-CoA pathway, which functions not only in acetyl-CoA assimilation. In Streptomyces sp., it may also supply precursors (ethylmalonyl-CoA) for antibiotic biosynthesis. For methylotrophic bacteria such as Methylobacterium extorquens, extension of the serine cycle with reactions of the ethylmalonyl-CoA pathway leads to a simplified scheme for isocitrate lyase-independent C1 assimilation.acetyl-CoA assimilation ͉ glyoxylate cycle ͉ methylotrophy ͉ polyketide ͉ serine cycle

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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

The metal-centered Δ/Λ-chirality of four-coordinated, nonplanar Zn(A(^)B)(2) complexes is correlated to the chirality of the bidentate enantiopure (R)-A(^)B or (S)-A(^)B Schiff base building blocks [A(^)B = (R)- or (S)-N-(1-(4-X-phenyl)ethyl)salicylaldiminato-κ(2)N,O with X = OCH(3), Cl, Br]. In the solid-state the (R) ligand chirality induces a Λ-M configuration and the (S) ligand chirality quantitatively gives the Δ-M configuration upon crystallization as deduced from X-ray single crystal studies. The diastereoselections of the pseudotetrahedral zinc-Schiff base complexes in CDCl(3) solution were investigated by (1)H NMR and by vibrational circular dichroism (VCD) spectroscopy. The appearance of two signals for the Schiff-base -CH═N- imine proton in (1)H NMR indicates an equilibrium of both Δ- and Λ-diastereomers with a diastereomeric ratio of roughly 20:80% for all three ligands. VCD proved to be very sensitive to the metal-centered Δ/Λ-chirality because of a characteristic band representing coupled vibrations of the two ligand's C═N stretch modes. The absolute configuration was assigned on the basis of agreement in sign with theoretical VCD spectra from Density Functional Theory calculations.