RESUMO
Acetyl-CoA carboxylases (ACCs) are enzyme complexes generally composed of three catalytic domains and distributed in all organisms. In prokaryotes and plastids of most plants, these domains are encoded in distinct subunits forming heteromeric complexes. Distinctively, cytosolic ACCs from eukaryotes and plastids of graminaceous monocots, are organized in a single multidomain polypeptide. Until now, no multidomain ACCs had been discovered in bacteria. Here, we show that a putative multidomain ACC in Saccharopolyspora erythraea is encoded by the sace_4237 gene, representing the first prokaryotic ACC homodimeric multidomain complex described. The SACE_4237 complex has both acetyl-CoA and propionyl-CoA carboxylase activities. Importantly, we demonstrate that sace_4237 is essential for S. erythraea survival as determined by the construction of a sace_4237 conditional mutant. Altogether, our results show that this prokaryotic homodimeric multidomain ACC provides malonyl-CoA for de novo fatty acid biosynthesis. Furthermore, the data presented here suggests that evolution of these enzyme complexes, from single domain subunits to eukaryotic multidomain ACCs, occurred in bacteria through domain fusion.
Assuntos
Carbono-Carbono Ligases/metabolismo , Ácidos Graxos/biossíntese , Malonil Coenzima A/metabolismo , Saccharopolyspora/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carbono-Carbono Ligases/química , Carbono-Carbono Ligases/genética , Filogenia , Domínios Proteicos , Saccharopolyspora/genética , Saccharopolyspora/crescimento & desenvolvimentoRESUMO
The taxonomic position of a rhizosphere soil isolate, designated strain SR8.15T, was determined by using a polyphasic approach. Phylogenetic analysis based on an almost-complete 16S rRNA gene sequence of the strain showed that it formed a well-separated sub-branch within the radiation encompassing the genus Saccharopolyspora. Highest levels of 16S rRNA gene sequence similarity were found between strain SR8.15T and Saccharopolyspora shandongensis CGMCC 4.3530T (98.9%) and Saccharopolyspora spinosa DSM 44228T (98.5%). However, these strains shared low levels of DNA-DNA relatedness (<26%). Strain SR8.15T had chemical characteristics consistent with its classification in the genus Saccharopolyspora. It contained meso-diaminopimelic acid as the diagnostic diamino acid. Whole-cell hydrolysates contained arabinose and galactose. The diagnostic phospholipids were phosphatidylcholine, phosphatidylglycerol and phosphatidylinositol. The main menaquinone was MK-9(H4). No mycolic acid was detected. The predominant cellular fatty acid was iso-C16:0. The G+C content of the genomic DNA of strain SR8.15T was 70.3 mol%. Strain SR8.15T had a phenotypic profile that readily distinguished it from recognized representatives of the genus Saccharopolyspora. It is evident from its combined genotypic and phenotypic properties that strain SR8.15T represents a novel species of the genus Saccharopolyspora, for which the name Saccharopolyspora phatthalungensis sp. nov. is proposed. The type strain is SR8.15T (=TISTR 1921T=BCC 35844T=NRRL B-24798T).
Assuntos
Hevea/microbiologia , Saccharopolyspora/classificação , Saccharopolyspora/isolamento & purificação , Microbiologia do Solo , DNA Bacteriano/genética , DNA Ribossômico/genética , Ácidos Graxos/metabolismo , Dados de Sequência Molecular , Filogenia , Raízes de Plantas/microbiologia , RNA Ribossômico 16S/genética , Saccharopolyspora/genética , Saccharopolyspora/metabolismoRESUMO
NADP+-Isocitrate dehydrogenase (ICDH) activity was detected in cell-free extracts of Saccharopolyspora erythraea CA340, an erythromycin producer. Apparent Km values for DL-isocitrate and NADP+ were 0.14 microM and 0.026 microM, respectively. ATP, ADP, GTP, citric acid, oxaloacetate, alpha-ketoglutarate, glyoxalate and glyoxalate plus oxaloacetate, each at 1 mM concentration, caused 50, 20 10, 50, 25, 60, 20 and 50% inhibition of ICDH activity, respectively. Phosphoenolpyruvate, fructose 1,6-diphosphate and pyruvate had no effect. ICDH specific activity profile was growth-associated and activity with dextrose or fructose as sole carbon source, was twice of that obtained with lactose.