Molecular, genetic, physiological and biochemical studies of 3-methylcrotonyl-CoA carboxylase and biotin carboxyl carrier protein-like proteins in Arabidopsis thaliana

Biotin-containing enzymes are involved in metabolic reactions that are essential for normal cellular functions in all organisms. In plants, four biotin-containing enzymes have been identified to date: homomeric acetyl-CoA carboxylase, heteromeric acetyl-CoA carboxylase, 3-methylcrotonyl-CoA carboxylase (MCCase), and geranoyl-CoA carboxylase. This dissertation contains two studies that are both part of the biotin metabolic network. The first study is the functional reverse genetic analysis of MCCase in Arabidopsis. MCCase is a nuclear-encoded, mitochondrial-localized enzyme. The reaction catalyzed by this enzyme is required for leucine (Leu) catabolism in mitochondria, and it may also play a role in the catabolism of isoprenoids and in the mevalonate shunt. In Arabidopsis, two MCCase subunits (the biotinylated MCCA subunit and the non-biotinylated MCCB subunit) are each encoded by single genes. To dissect the physiological role of MCCase in plants Arabidopsis MCCase mutants were isolated and characterized. Metabolite profiling studies indicate that mutations in either MCCA or MCCB block mitochondrial Leu catabolism. Under light deprivation conditions, the hyper accumulation of Leu, 3-methylcrotonyl-CoA and isovaleryl-CoA suggests that two Leu catabolism pathway, i.e., the mitochondrial and the peroxisomal Leu catabolism pathways, are independently regulated. The biochemical block in mitochondrial Leu catabolism is associated with an impaired reproductive growth phenotype, including aberrant flower and silique development, and decreased seed germination. The decreased seed germination phenotype is only expressed when homozygous mutant seeds are collected from a parent plant that is itself homozygous, but not when collected from a parent plant that is heterozygous. These observations indicate that maternal mitochondrial Leu catabolism is required for normal seed development. The second study is the characterization of three Arabidopsis genes that code for biotin carboxyl carrier protein-like (BCCPL) proteins. BCCPL proteins were first identified using bioinformatic approaches. We hypothesized that these proteins may represent novel biotin- or lipoylate-containing proteins in plants. Immunological analysis demonstrates that BCCPL proteins are indeed expressed in plants. However, affinity chromatography purification indicates that these proteins are not biotinylated, nor lipoylated in plants. T-DNA knockout alleles of BCCPL genes were recovered and characterized, yet no observable phenotypic changes were found. The biochemical and physiological functions of BCCPLs remains unclear.