Biochemical and molecular genetic studies of the metabolic role of methylcrotonyl-CoA carboxylase
The physiological metabolic roles of methylcrotonyl-CoA carboxylase (MCCase) have been investigated. Radiotracer metabolic studies using extracts from isolated mitochondria and incubated with [U-14C]leucine and NaH14CO3, demonstrate that plant mitochondria can catabolize leucine via the following scheme: leucine ⇒ alpha-ketoisocaproate ⇒ isovaleryl-CoA ⇒ 3-methylcrotonyl-CoA ⇒ 3-methylglutaconyl-CoA ⇒ 3-hydroxy-3-methylglutaryl-CoA ⇒ acetoacetate + acetyl-CoA. These findings demonstrate for the first time that the enzymes responsible for leucine catabolism are present in plant mitochondria. We conclude that one of the primary metabolic roles of MCCase in plants is the catabolism of leucine;A single-copy gene encoding the Arabidopsis MCC-A subunit was isolated and characterized. It contains 15 exons separated by 14 introns. The expression of the Arabidopsis MCC-A and MCC-B genes were examined using transgenic expression of beta-glucuronidase (GUS) reporter gene fused to each respective promoter sequence. The light mediated repression of MCCase expression reveals that the expression of MCC-A and MCC-B genes are under metabolic control at the transcriptional level. This mechanism was tested by artificially depriving carbohydrate biosynthesis (by placing plants in darkness and CO2 free atmosphere) and by blocking carbohydrate translocation to roots (by detaching leaves exogenous sugars reverse these effects. Our studies also demonstrated that, in all cases, the expressions of MCC-A and MCC-B genes are coordinately regulated;Biotin plays multiple roles during the growth and development of an organism. Our studies showed for the first time that MCCase is down-regulated by biotin at the translational and/or post-translational level. Although biotin had no effects on MCCase gene transcription, it is required for the MCCase response to plant metabolic changes at the transcriptional level;Comparison of the molecular weight of MCCase reveals that different species have different MCCase subunit stochiometry. We determined herein that the subunit composition of Arabidopsis MCCase is A4B 4. Biotinylation has no effect on this subunit stochiometry.