PROPIONYL-COA CARBOXYLASE FROM STREPTOMYCES-COELICOLOR A3(2) - CLONING OF THE GENE ENCODING THE BIOTIN-CONTAINING SUBUNIT

In Streptomyces coelicolor A3(2), polyketides are made from malonyl-Co A, which is presumed to be derived from acetyl-CoA by the action of ac etyl-CoA carboxylase (ACC). No ACC activity was found in cell-free ext racts of S. coelicolor. However, propionyl-CoA carboxylase (PCC) activ ity was detected at substantial levels. Fixation of CO2 by ACC and PCC occurs by covalent bonding of CO2 to a biotin-containing protein. Mos t bacteria have a single small biotinylated protein of approximately 2 2 kDa, but S. coelicolor contains three larger biotin-containing prote ins (approximately 145, 88 and 70 kDa). To determine which biotinylate d protein was associated with PCC activity, the enzyme was purified an d shown to comprise an ct subunit (biotin-containing) of 88 kDa and a beta subunit of 66 kDa. The N-terminal sequences of these proteins wer e determined and, using an oligonucleotide probe, the gene for the a s ubunit (pccA) was cloned.