The enzymic stages of mammalian mitochondrial β-oxidation were elucidated some 30–40 years ago. However, the discovery of a membrane-associated multifunctional enzyme of β-oxidation, a membrane-associated acyl-CoA dehydrogenase and characterization of the carnitine palmitoyl transferase system at the protein and at the genetic level has demonstrated that the enzymes of the system itself are incompletely understood. Deficiencies of many of the enzymes have been recognized as important causes of disease. In addition, the study of these disorders has led to a greater understanding of the molecular mechanism of β-oxidation and the import, processing and assembly of the β-oxidation enzymes within the mitochondrion. The tissue-specific regulation, intramitochondrial control and supramolecular organization of the pathway is becoming better understood as sensitive analytical and molecular techniques are applied. This review aims to cover enzymological and organizational aspects of mitochondrial β-oxidation together with the biochemical aspects of inherited disorders of β-oxidation and the intrinsic control of β-oxidation.

Multiple acyl-CoA dehydrogenation deficiency (MADD) is a disorder of fatty acid, amino acid and choline metabolism that can result from defects in two flavoproteins, electron transfer flavoprotein (ETF) or ETF: ubiquinone oxidoreductase (ETF:QO). Some patients respond to pharmacological doses of riboflavin. It is unknown whether these patients have defects in the flavoproteins themselves or defects in the formation of the cofactor, FAD, from riboflavin. We report 15 patients from 11 pedigrees. All the index cases presented with encephalopathy or muscle weakness or a combination of these symptoms; several had previously suffered cyclical vomiting. Urine organic acid and plasma acyl-carnitine profiles indicated MADD. Clinical and biochemical parameters were either totally or partly corrected after riboflavin treatment. All patients had mutations in the gene for ETF:QO. In one patient, we show that the ETF:QO mutations are associated with a riboflavin-sensitive impairment of ETF:QO activity. This patient also had partial deficiencies of flavin-dependent acyl-CoA dehydrogenases and respiratory chain complexes, most of which were restored to control levels after riboflavin treatment. Low activities of mitochondrial flavoproteins or respiratory chain complexes have been reported previously in two of our patients with ETF:QO mutations. We postulate that riboflavin-responsive MADD may result from defects of ETF:QO combined with general mitochondrial dysfunction. This is the largest collection of riboflavin-responsive MADD patients ever reported, and the first demonstration of the molecular genetic basis for the disorder.

A young girl presented with recurrent episodes of muscle weakness culminating in a severe attack of generalized muscle weakness. In the muscle mitochondria from the patient there was an abnormal pattern of intermediates of beta-oxidation with an accumulation of 3-hydroxyacyl- and 2-enoyl-CoA and carnitine esters, and 3-oxoacylcarnitines. There was low activity of long-chain 3-hydroxyacyl-CoA dehydrogenase in mitochondria from all tissues. The activity of long-chain 2-enoyl-CoA hydratase was low in muscle mitochondria and 3-oxoacyl-CoA thiolase activity measured with 3-oxohexadecanoyl-CoA as substrate was low in fibroblast, muscle, and cardiac mitochondria but only partial deficiency was present when the activity was measured with 3-oxooctanoyl-CoA. The activity of the long-chain 3-hydroxyacyl-CoA dehydrogenase and long-chain 3-oxoacyl-CoA thiolase in fibroblasts from the patient's parents was intermediate between those of controls and the patient. The patient has a combined defect of the long-chain 3-hydroxyacyl-CoA dehydrogenase, long-chain 3-oxoacyl-CoA thiolase, and long-chain 2-enoyl-CoA hydratase which appears to be inherited in an autosomal recessive manner. This suggests there is a multifunctional enzyme catalyzing these activities in human mitochondria and that this enzyme is deficient in our patient.

A young man presented with recurrent episodes of muscle pain and myoglobinuria after prolonged exercise or fasting. Studies on isolated muscle mitochondria showed slow flux through β-oxidation and the presence of only saturated long-chain acyl coenzyme A (acyl-CoA) esters. These results strongly suggested a defect in the dehydrogenation of long-chain acyl-CoA esters that we confirmed by measurement of enzyme activity in muscle and platelet mitochondrial fractions and fibroblast homogenates. In all tissues studied from the patient, the enzyme activity was approximately 10% of control values with acyl-CoA esters from C₁₆-C₂₂ as substrates. We investigated the intramito-chondrial location of the deficient acyl-CoA dehydrogenase by subfractionation of platelet mitochondria and, in contrast to the short-chain and medium-chain enzymes, which were localized in the soluble fraction, the majority of the acyl-CoA dehydrogenase activity with long-chain substrates was in the membrane fraction. These studies indicate that in humans, the predominant enzyme catalyzing the dehydrogenation of long-chain acyl-CoA esters is membrane-bound and that deficiency of this enzyme is a cause of muscle pain and rhabdomyolysis.

The trifunctional enzyme comprises three consecutive steps in the mitochondrial β-oxidation of long-chain acyl-CoA esters: 2-enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase. Deficiencies in either 3-hydroxyacyl-CoA dehydrogenase activity, or all three activities, are important causes of human disease. The dehydrogenase and thiolase have a requirement for NAD+ and CoA respectively, whose levels are conserved within the mitochondrion and thus provide possible means for control and regulation of β-oxidation. Using analysis of the intact CoA ester intermediates produced by the complex, we have examined the sensitivity of the complex to NAD+/NADH and acetyl-CoA. We consider the evidence for channelling within the trifunctional protein and propose a model for a β-oxidation ‘metabolon’.