MOLECULAR-CLONING AND CHARACTERIZATION OF THE CDNA CODING FOR THE BIOTIN-CONTAINING SUBUNIT OF 3-METHYLCROTONOYL-COA CARBOXYLASE - IDENTIFICATION OF THE BIOTIN CARBOXYLASE AND BIOTIN-CARRIER DOMAINS

Soybean genomic clones were isolated based on hybridization to probes that code for the conserved biotinylation domain of biotin-containing enzymes, The corresponding cDNA was isolated and expressed in Escheric hia coli through fusion to the bacterial trpE gene. The resulting chim eric protein was biotinylated in E. coli. Antibodies raised against th e chimeric protein reacted specifically with an 85-kDa biotin-containi ng polypeptide from soybean and inhibited 3-methylcrotonoyl-CoA carbox ylase (EC 6.4.1.4) activity in cell-free extracts of soybean leaves. T hus, the isolated soybean gene and corresponding cDNA code for the 85- kDa biotin-containing subunit of 3-methylcrotonoyl-CoA carboxylase. Th e nucleotide sequence of the cDNA and portions of the genomic clones w as determined. Comparison of the deduced amino acid sequence of the bi otin-containing subunit of 3-methylcrotonoyl-CoA carboxylase with sequ ences of other biotin enzymes suggests that this subunit contains the functional domains for the first half-reaction catalyzed by all biotin -dependent carboxylases-namely, the carboxylation of biotin. These dom ains are arranged serially on the polypeptide, with the biotin carboxy lase domain at the amino terminus and the biotin-carboxyl carrier doma in at the carboxyl terminus.