Kinetic and Structural Analysis of a New Group of Acyl-CoA Carboxylases Found in Streptomyces coelicolor A3(2)

Abstract

Two acyl-CoA carboxylases from Streptomyces coelicolor have been successfully reconstituted from their purified components. Both complexes shared the same biotinylated ␣ subunit, AccA2. The ␤ and the ⑀ subunits were specific from each of the complexes; thus, for the propionyl-CoA carboxylase complex the ␤ and ⑀ components are PccB and PccE, whereas for the acetyl-CoA carboxylase complex the components are AccB and AccE. The two complexes showed very low activity in the absence of the corresponding ⑀ subunits; addition of PccE or AccE dramatically increased the specific activity of the enzymes. The kinetic properties of the two acyl-CoA carboxylases showed a clear difference in their substrate specificity. The acetyl-CoA carboxylase was able to carboxylate acetyl-, propionyl-, or butyryl-CoA with approximately the same specificity. The propionylCoA carboxylase could not recognize acetyl-CoA as a substrate, whereas the specificity constant for propionyl-CoA was 2-fold higher than for butyryl-CoA. For both enzymes the ⑀ subunits were found to specifically interact with their carboxyltransferase component forming a ␤-⑀ subcomplex; this appears to facilitate the further interaction of these subunits with the ␣ component. The ⑀ subunit has been found genetically linked to several carboxyltransferases of different Streptomyces species; we propose that this subunit reflects a distinctive characteristic of a new group of acyl-CoA carboxylases.The first committed step in the biosynthesis of long-chain fatty acids in all animals, plants, and bacteria is catalyzed by acetyl-CoA carboxylase (ACC) 1 (EC 6.4.1.2) (1). The reaction catalyzed by ACC involves two separate reactions.

Biochemical and Structural Characterization of an Essential Acyl Coenzyme A Carboxylase from Mycobacterium tuberculosis

“…The production of ADP was coupled to pyruvate kinase and lactate dehydrogenase, and the oxidation of NADH was monitored at 340 nm. Assays were performed in a microplate reader as previously described (20). Data were collected using a Dynex MRX microplate reader interfaced to a personal computer equipped with a data acquisition program.…”

“…Consequently, these enzymes are referred to as acyl-CoA carboxylases (ACCase), and all of them consist of a large subunit (the ␣-chain) with the ability to carboxylate its covalently bound biotin group, and a smaller subunit (the ␤-chain) bearing the carboxyltransferase activity. In our laboratory, two ACCase complexes from Streptomyces coelicolor A3(2) have been widely characterized at both the biochemical and structural levels (19,20,46,47). As a new feature of this group of enzymes, we found a third subunit, called ε, which is essential for the maximal activity of the complexes (20).…”

“…In our laboratory, two ACCase complexes from Streptomyces coelicolor A3(2) have been widely characterized at both the biochemical and structural levels (19,20,46,47). As a new feature of this group of enzymes, we found a third subunit, called ε, which is essential for the maximal activity of the complexes (20).…”

Complete genome sequence of the erythromycin-producing bacterium Saccharopolyspora erythraea NRRL23338

“…3), and in S. erythraea it appears that there are at least five genetic loci, displaying remarkably diverse protein architectures, that might code for an enzyme catalyzing this reaction ( Supplementary Fig. 3 online) 35,36 . Further work will be required to deconvolute the contributions made by these gene sets to erythromycin biosynthesis.…”

Complete genome sequence of the rifamycin SV-producing Amycolatopsis mediterranei U32 revealed its genetic characteristics in phylogeny and metabolism

“…There are at least four sets of genes encoding the acetyl-CoA and propionyl-CoA carboxylase complexes (Supplementary information, Table S1), which are used to provide malonyl-CoA and (S)-methylmalonyl-CoA for the synthesis of fatty acids and polyketides, particularly rifamycin [1,39]. An alternative pathway to generate (S)-methylmalonylCoA is catalyzed by methylmalonyl-CoA mutases (Supplementary information, Table S1), converting succinylCoA to (R)-methylmalonyl-CoA and then to (S)-isomers by methylmalonyl-CoA epimerase [40].…”