Molecular cloning and characterization of 3-methylcrotonyl-CoA carboxylase from soybean

Song, Jianping
Major Professor
Basil J. Nikolau
Committee Member
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Biochemistry, Biophysics and Molecular Biology

A cDNA clone and genes coding for the 85 kDa biotin-containing polypeptide of soybean has been isolated and sequenced. This 85 kDa polypeptide has been identified as the biotin-containing subunit of 3-methylcrotonyl-CoA carboxylase (MCCase). This is the first plant gene coding for a biotin-containing enzyme to be cloned. It is also the first time that the primary sequence of the biotin-containing subunit of MCCase has been determined;The deduced amino acid sequence of the biotin-containing subunit of MCCase of soybean shows high homology with other known biotin-containing enzymes. Amino acid sequence homology between the soybean MCCase and other biotin enzymes identified the biotin carboxylase and the biotin carboxyl carrier domains. In addition, the first 21 amino acid residues have the characteristics of a mitochondrial targeting signal peptide. The cleavage site of the signal peptide of the soybean biotin-containing subunit of MCCase has been confirmed by the direct sequencing of the N-terminus of the mature protein;Soybean MCCase has been purified to near homogeneity by a five step purification procedure. The purified MCCase is composed of equal proportions of a 85 kDa biotin-containing subunit and a 59 kDa biotin-free subunit, with a molecular weight of approximately 970,000 ± 83,000. Soybean MCCase appears to have an [alpha][subscript]6[beta][subscript]6 quaternary structure;Initial velocity kinetic studies of soybean MCCase with competitive inhibitors demonstrated that soybean MCCase follows a random Bi Bi Uni Uni Ping Pong mechanism;Putative isozymes of the soybean MCCase have been discovered for the first time. Isoform I is more active and has a lower K[subscript] m for ATP compared to isoform II. The K[subscript] ms for HCO[subscript]3[superscript]- and 3-methylcrotonyl-CoA (Mc-CoA) are similar for both forms. The molecular weight of the native isoforms are the same, and they possess biotin-containing subunits of identical molecular weights;The effect of development on MCCase activity and accumulation of the biotin subunit and mRNA coding for this subunit have been examined. The results are consistent with the presumed role of this enzyme in leucine catabolism, namely, MCCase is highly expressed in tissues that need to generate ATP and reducing equivalents via respiration of organic constituents.