Synthesis of C ₅ -dicarboxylic acids from C ₂ -units involving crotonyl-CoA carboxylase/reductase: The ethylmalonyl-CoA pathway

Abstract

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

Mining the Cinnabaramide Biosynthetic Pathway to Generate Novel Proteasome Inhibitors

“…In this pathway, reductive carboxylation of (E)-crotonylCoA by a crotonyl-CoA carboxylase/reductase (Ccr) enzyme [30] with use of the purified enzyme in vitro has been described. Similar biochemical steps have also been reported to be involved in the production of the PKS extender units ethylmalonyl-CoA and chloroethylmalonyl-CoA in the salinosporamide biosynthesis pathway.…”

Study of the distribution of autotrophic CO2 fixation cycles in Crenarchaeota

“…The reaction was stopped after different time intervals by mixing the samples (50 ml) with 10 ml 1 M HCl. The samples were centrifuged (4 uC, 20 000 g, 15 min) and analysed by HPLC using an RP-C 18 column, as described previously (Erb et al, 2007). The identification of the CoA esters was based on co-chromatography with standards (Erb et al, 2007;Berg et al, 2007).…”

“…In S. azoricus and M. sedula, 4-hydroxybutyryl-CoA dehydratase (EC 4.2.1.-) activity was measured spectrophotometrically at 42 uC in an assay coupled with the recombinant crotonyl-CoA carboxylase/ reductase from Rhodobacter sphaeroides (Erb et al, 2007), as described previously . Since the growth temperature of P. fumarii is 20 uC higher than that of S. azoricus, a discontinuous assay was used in this case (Ramos-Vera et al, 2009).…”

The methylaspartate cycle in haloarchaea and its possible role in carbon metabolism

“…Interestingly, the ethylmalonyl-CoA pathway, another alternative to the glyoxylate cycle functioning in various proteobacteria and streptomycetes (Erb et al, 2007(Erb et al, , 2009, involves formation of acetoacetyl-CoA, that is also an intermediate of polyhydroxybutyrate biosynthesis and degradation (Dawes, 1988). Like the methylaspartate cycle, the ethylmalonyl-CoA pathway involves the intermediates mesaconyl-CoA and methylmalyl-CoA and both pathways result in the formation and assimilation of propionyl-CoA.…”