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Microbiology 145 (1999), 3109-3119
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Microbiology (1999), 145, 3109-3119.
© 1999 Society for General Microbiology

Genetics and Molecular Biology

Genetic and biochemical characterization of the {alpha} and ß components of a propionyl-CoA carboxylase complex of Streptomyces coelicolor A3(2)

E. Rodríguez1 and H. Gramajo1

Programa Multidisciplinario de Biología Experimental (PROMUBIE-CONICET) and Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, 2000-Rosario, Argentina1

Author for correspondence: H. Gramajo. Tel: +54 341 4350661. Fax: +54 341 4390465. e-mail: gramajo{at}infovia.com.ar

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 coli and in vitro reconstitution of enzyme activity confirmed that PccB is the ß 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.

Keywords: acyl-CoA carboxylase, primary metabolism, propionyl-CoA carboxylase, biotinylated protein

Abbreviations: ACCase, acetyl-CoA carboxylase; Am, apramycin; Ap, ampicillin; BC, biotin carboxylase; BCCP, biotin-carboxyl carrier protein; Cm, chloramphenicol; Km, kanamycin; mmCoA, methylmalonyl-CoA; PCCase, propionyl-CoA carboxylase; Th, thiostrepton

The GenBank accession numbers for the accA1, aacA2 and pccB sequences determined in this work are AF113603, AF113604 and AF113605, respectively.




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