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

Abstract

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 Escherichia coli through fusion to the bacterial trpE gene. The resulting chimeric protein was biotinylated in E. coli. Antibodies raised against the chimeric protein reacted specifically with an 85-kDa biotin-containing polypeptide from soybean and inhibited 3-methylcrotonoyl-CoA carboxylase (EC 6.4.1.4) activity in cell-free extracts of soybean leaves. Thus, the isolated soybean gene and corresponding cDNA code for the 85-kDa biotin-containing subunit of 3-methylcrotonoyl-CoA carboxylase. The nucleotide sequence of the cDNA and portions of the genomic clones was determined. Comparison of the deduced amino acid sequence of the biotin-containing subunit of 3-methylcrotonoyl-CoA carboxylase with sequences 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 domains are arranged serially on the polypeptide, with the biotin carboxylase domain at the amino terminus and the biotin-carboxyl carrier domain at the carboxyl terminus.