Genetic and biochemical characterization of the alpha and beta components of a propionyl-CoA carboxylase complex of Streptomyces coelicolor A3(2).
Two genes, accA1 and accA2, with nearly identical nucleotide sequences were cloned from Streptomyces coelicolor A3(2). The deduced amino acid sequences of the product of these two genes showed high similarity to BcpA2 of Saccharopolyspora erythraea and other biotin-containing proteins from different organisms assumed to be the alpha subunit of a propionyl-CoA carboxylase. A gene, pccB, encoding the carboxyl transferase subunit of this enzyme complex was also characterized. Strains disrupted in accA1 did not show any change in acetyl- or propionyl-CoA carboxylase activity, whilst cell-free extracts of a pccB mutant strain contained a reduced level of propionyl-CoA carboxylase. No mutants in accA2 could be isolated, suggesting that the gene may be essential. Heterologous expression of accA1, accA2 and pccB in Escherichia col and in vitro reconstitution of enzyme activity confirmed that PccB is the beta subunit of a propionyl-CoA carboxylase and that either AccA1 or AccA2 could act as the alpha component of this enzyme complex. The fact that accA2 mutants appear to be inviable suggests that this gene encodes a biotinylated protein that might be shared with other carboxyl transferases essential for the growth of S. coelicolor.
Rodrı Guez
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E
Programa Multidisciplinario de Biologı́a Experimental (PROMUBIE-CONICET) and Departamento de Microbiologı́a, Facultad de Ciencias Bioquı́micas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, 2000-Rosario, Argentina1.
Gramajo
H
H
Programa Multidisciplinario de Biologı́a Experimental (PROMUBIE-CONICET) and Departamento de Microbiologı́a, Facultad de Ciencias Bioquı́micas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, 2000-Rosario, Argentina1.
D001483:Base Sequence; D019921:Biotinylation; D015153:Blotting, Western; D019735:Carbon-Carbon Ligases / Q000235:genetics; D002262:Carboxy-Lyases / Q000235:genetics* / Q000378:metabolism; D019878:Carboxyl and Carbamoyl Transferases / Q000235:genetics; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D043586:Methylmalonyl-CoA Decarboxylase; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics*; D014169:Transformation, Bacterial
D011994:Recombinant Proteins; D019878:Carboxyl and Carbamoyl Transferases; D002262:Carboxy-Lyases; D019735:Carbon-Carbon Ligases; C018757:acyl-CoA carboxylase; D043586:Methylmalonyl-CoA Decarboxylase