Typewritten copy Includes bibliographical references.

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The 2-methylbranched chain acyl-CoA dehydrogenase (BCAD) is a mitochondrial enzyme that catalyzes the third reaction in isoleucine and valine metabolism, the oxidation of 2-methylbutyryl-CoA and isobutyryl-CoA, respectively. BCAD deficiency would result in the accumulation of branched chain acyl-CoAs or their derivatives. Three patients with a putative defect in BCAD have been reported. This study consists of a molecular examination of one such patient as well as the characterization of the BCAD gene. In Northern blot analysis of human fibroblast RNA, the BCAD cDNA hybridized to two RNA species of 2.7 and 6.5 kb. The 2.7 kb band corresponds to the size of the BCAD cDNA, which consists of the entire coding region of 1.3 kb and a 3$ sp prime$ untranslated region of 1.4 kb. The coding regions of the BCAD gene span approximately 21 kb and consist of 12 exons and 11 introns. The exons range in size from 39 to 108 bp. In the analysis of the putative BCAD-deficient patient, no significant difference was observed at the level of DNA (Southern), RNA (Northern) or protein (Western) when compared to controls, suggesting that the BCAD gene in this patient did not contain any large insertions or deletions, or a frameshift mutation. The single strand conformation polymorphism (SSCP) technique and sequencing of the entire coding region did not reveal any disease-causing mutations but two polymorphisms were identified: one in exon 6 and the other in exon 10.

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Biotin is a water soluble vitamin that is mainly used as a cofactor in carboxylation reactions by a class of enzyme known as biotin-dependent carboxylases. In order to act as a cofactor, the biotin molecule has to be covalently attached to a lysine residue by an enzyme called holocarboxylase synthetase (HCS). Inherited deficiency of the biotin-dependent propionyl-CoA carboxylase (PCC) results in the inborn error of metabolism propionic acidemia. Mutations in either the alpha (PCCA gene) or beta (PCCB gene) subunit of the enzyme have been shown to cause propionic acidemia. Mutation analysis of the PCCB gene have revealed several mutations. However, few PCCalpha mutations have been described. The first goal of this thesis was to determine the molecular etiology of alpha subunit deficiency at the mRNA as well as at the protein level. I found that most mutations destabilized either the mRNA or the protein. Two other mutations were found to affect the biotinylation of PCCalpha, defining residues important for the folding of the domain or for interaction with HCS. The second part of my thesis was to study in more details the interactions between HCS and the biotinylation domain of PCCalpha, represented by the last 67 amino acids of the subunit (p-67). I expressed and purified p-67 from Pichia pastoris. I compared p-67 with the E. coli biotinylation domain (BCCP87) as substrates for the E. coli orthologous enzyme BirA, using steady-state as well as stopped-flow kinetics. I noticed some differences between these two substrates and how it might relate to the biotinylation reaction. I generated N-terminal and C-terminal deletions of HCS and I tested their activity in vivo and in vitro using purified susbtrates. I was able to map the minimal sequence requirement for HCS activity to the last 348 amino acids of the enzyme. I also found that some longer HCS were either almost or totally inactive or some that were active showed a differential activity towards the different susb

Short/branched chain acyl-CoA dehydrogenase (SBCAD), a member of the acyl-CoA dehydrogenase (ACD) family of enzymes, catalyzes the oxidation of branched chain fatty acids and the branched chain amino acids isoleucine and valine. This research project focuses on expression studies of the SBCAD gene. Northern blot analysis detected two SBCAD mRNA species of 2.7 and 6.5 kb in various human tissues and cell types. A single 4.1 and 2.0 kb SBCAD message was detected in rat and pig tissues, respectively, revealing a species difference in SBCAD mRNA size. Studies of human and rat SBCAD tissue-specificity and relative abundance, at both the RNA and protein levels, identified liver and kidney as the tissues with the highest levels of SBCAD expression, establishing a unique tissue-specific expression pattern that is not seen among the other members of the ACD family. Furthermore, a fetal and adult difference in SBCAD expression was observed in human kidney, suggesting that the SBCAD gene may be developmentally regulated in some tissues. Finally, an attempt was made to isolate and characterize the SBCAD promoter region in order to provide valuable data for future SBCAD promoter studies.

The objective of this thesis was to determine the molecular basis of neonatal multiple carboxylase deficiency (MCD) produced by an impairment in holocarboxylase synthetase (HCS) activity and the origin of the biotin-responsiveness that characterizes this disease. To determine HCS activity, I developed a peptide substrate and used the biotinylation system of E: coli to determine its properties. C-terminal fragments of the $ alpha$ subunit of human propionyl-CoA carboxylase (PCC-$ alpha$) were expressed in E. coli and site-directed mutagenesis was used to define the residues required for biotinylation by the bacterial biotin ligase, BirA. These experiments showed that the biotin region of PCC-$ alpha$ can act as an autonomous domain for biotinylation and suggested its use as substrate for human HCS. For the molecular characterization of MCD, I isolated several cDNA clones encoding human HCS by functional complementation of an E. coli mutant with a temperature-sensitive BirA. Comparison of the predicted amino acid sequence of HCS with bacterial biotin ligases allowed the identification of the putative biotin-binding domain of this protein. Mutation analysis of DNA from HCS deficient patients showed that most of the changes in the HCS sequence are clustered in the biotin-binding domain. All the patients tested in this study showed deficiency of HCS activity as determined using the PCC-$ alpha$ peptide as substrate for biotinylation. The biotin-responsiveness was demonstrated by obtaining a stimulation of HCS activity of MCD cells at high biotin concentrations while remaining unstimulated in extracts of normal cells. Together with the mutation studies, these results showed that neonatal MCD is caused by mutations in the biotin binding domain of HCS which reduce the affinity of the enzyme towards biotin. This change in the kinetic properties of HCS results in the inefficient biotinylation of carboxylases at physiological concentrations of biotin. The defect can be over

Multiple carboxylase deficiency (MCD) results from a decreased activity of holocarboxylase synthetase (HCS) which is responsible for the biotinylation of the four biotin-dependent carboxylases found in humans. The disease can be treated with pharmacologic doses of oral biotin (biotin-responsiveness). The cDNA for HCS contains a biotin-binding domain deduced by analogy with the sequence and crystal structure of the E. coli BirA biotin ligase. E. coli birA$ sp-$ mutations causing biotin-auxotrophy all localize to this region. Of six point mutations I have identified in MCD patients, four localize to the biotin-binding region. In order to assess the HCS activity associated with patient mutations, I used an assay based on the expression of mutant HCS in E. coli. The method is based on the ability of mutant HCS to biotinylate the biotin carboxyl carrier protein (BCCP) of acetyl-CoA carboxylase in a temperature-sensitive birA$ sp-$ E. coli strain using 3H-biotin as tracer. I have shown that all of the mutations cause a severe decrease in HCS activity. In addition, I have shown that five of the mutant HCS are biotin-responsive. These findings are a major contribution to the understanding of the mechanism of biotin-responsiveness.