Molecular characterization of the non-biotin-containing subunit of 3-methylcrotonyl-CoA carboxylase

The biotin enzyme, 3-methylcrotonyl-CoA carboxylase (MCCase) (3-methylcroto nyl-CoA:carbon-dioxide ligase (ADP-forming), EC 6.4.1.4), catalyzes a pivot al reaction required for both leucine catabolism and isoprenoid metabolism. MCCase is a heteromeric enzyme composed of biotin-containing (MCC-A) and n on-biotin-containing (MCC-B) subunits. Although the sequence of the MCC-A s ubunit was previously determined, the primary structure of the MCC-B subuni t is unknown. Based upon sequences of biotin enzymes that use substrates st ructurally related to 3-methylcrotonyl-CoA, we isolated the MCC-B cDNA and gene of Arabidopsis. Antibodies directed against the bacterially produced r ecombinant protein encoded by the MCC-B cDNA react solely with the MCC-B su bunit of the purified MCCase and inhibit MCCase activity. The primary struc ture of the MCC-B subunit shows the highest similarity to carboxyltransfera se domains of biotin enzymes that use methyl-branched thiol esters as subst rate or products. The single copy MCC-B gene of Arabidopsis is interrupted by nine introns. MCC-A and MCC-B mRNAs accumulate in all cell types and org ans, with the highest accumulation occurring in rapidly growing and metabol ically active tissues. In addition, these two mRNAs accumulate coordinately in an approximately equal molar ratio, and they each account for between 0 .01 and 0.1 mol ac of cellular mRNA The sequence of the Arabidopsis MCC-B g ene has enabled the identification of animal paralogous MCC-B cDNAs and gen es, which may have an impact on the molecular understanding of the lethal i nherited metabolic disorder methylcrotonylglyciuria.