Cloning and characterization of a human cDNA ACAD10 mapped to chromosome 12q24.1

Mitochondrial fatty acid -oxidation is an important energy resource for many mammal tissues. Acyl-CoA dehydrogenases (ACADs) are a family of flavoproteins that are involved in the -oxidation of the fatty acyl-CoA derivatives. Deficiency of these ACADs can cause metabolic disorders including muscle fatigue, hypoglycaemia, hepatic lipidosis and so on. By large scale sequencing, we identified a cDNA sequence of 3960 base pairs with a typical acyl-CoA dehydrogenase function domain. RT-PCR result shows that it is widely expressed in human tissues, especially high in liver, kidney, pancreas and spleen. It is hypothesized that this is a novel member of ACADs family. Abbreviations: ACADs – acyl-CoA dehydrogenases, FAD – flavinadenine dinucleotide, SCAD – short-chain acyl-CoA dehydrogenase,MCAD – medium-chain acyl-CoA dehydrogenase, LCAD – long-chain acyl-CoAdehydrogenase, VLCAD – very long- chain acyl-CoA dehydrogenase, IVD –isocalery-CoA dehydrogenase, SBCAD – short/branched chain acyl-CoAdehydrogenase, GCD – glutaryl- CoA dehydrogenase, ETF – electron transferflavoprotein, ACAD8 – acyl-CoA dehydrogenase 8, ACAD9 – acyl-CoAdehydrogenase 9, ACAD10 – acyl-CoA dehydrogenase 10.     

Crystal structure of rat short chain acyl-CoA dehydrogenase complexed with acetoacetyl-CoA: comparison with other acyl-CoA dehydrogenases.

The acyl-CoA dehydrogenases are a family of flavin adenine dinucleotide-containing enzymes that catalyze the first step in the beta-oxidation of fatty acids and catabolism of some amino acids. They exhibit high sequence identity and yet are quite specific in their substrate binding. Short chain acyl-CoA dehydrogenase has maximal activity toward butyryl-CoA and negligible activity toward substrates longer than octanoyl-CoA. The crystal structure of rat short chain acyl-CoA dehydrogenase complexed with the inhibitor acetoacetyl-CoA has been determined at 2.25 A resolution. Short chain acyl-CoA dehydrogenase is a homotetramer with a subunit mass of 43 kDa and crystallizes in the space group P321 with a = 143.61 A and c = 77.46 A. There are two monomers in the asymmetric unit. The overall structure of short chain acyl-CoA dehydrogenase is very similar to those of medium chain acyl-CoA dehydrogenase, isovaleryl-CoA dehydrogenase, and bacterial short chain acyl-CoA dehydrogenase with a three-domain structure composed of N- and C-terminal alpha-helical domains separated by a beta-sheet domain. Comparison to other acyl-CoA dehydrogenases has provided additional insight into the basis of substrate specificity and the nature of the oxidase activity in this enzyme family. Ten reported pathogenic human mutations and two polymorphisms have been mapped onto the structure of short chain acyl-CoA dehydrogenase. None of the mutations directly affect the binding cavity or intersubunit interactions.     

Mammalian electron transferring flavoprotein.flavoprotein dehydrogenase complexes observed by microelectrospray ionization-mass spectrometry and surface plasmon resonance

Microelectrospray ionization-mass spectrometry was used to directly observe electron transferring flavoprotein.flavoprotein dehydrogenase interactions. When electron transferring flavoprotein and porcine dimethylglycine dehydrogenase or sarcosine dehydrogenase were incubated together in the absence of substrate, a relative molecular mass corresponding to the flavoprotein.electron transferring flavoprotein complex was observed, providing the first direct observation of these mammalian complexes. When an acyl-CoA dehydrogenase family member, human short chain acyl-CoA dehydrogenase, was incubated with dimethylglycine dehydrogenase and electron transferring flavoprotein, the microelectrospray ionization-mass spectrometry signal for the dimethylglycine dehydrogenase.electron transferring flavoprotein complex decreased, indicating that the acyl-CoA dehydrogenases have the ability to compete with the dimethylglycine dehydrogenase/sarcosine dehydrogenase family for access to electron transferring flavoprotein. Surface plasmon resonance solution competition experiments revealed affinity constants of 2.0 and 5.0 microm for the dimethylglycine dehydrogenase-electron transferring flavoprotein and short chain acyl-CoA dehydrogenase-electron transferring flavoprotein interactions, respectively, suggesting the same or closely overlapping binding motif(s) on electron transferring flavoprotein for dehydrogenase interaction.     

Effects of branched chain alpha-ketoacids on the metabolism of isolated rat liver cells. I. Regulation of branched chain alpha-ketoacid metabolism.

alpha-Ketoisocaproate (ketoleucine) is shown to be metabolized to ketone bodies rapidly by isolated rat liver cells. Acetoacetate is the major end product and maximum rates were observed with 2 mM substrate. Studies with 2-tetradecylglycidic acid (an inhibitor of long chain fatty acid oxidation) showed that ketogenesis from alpha-ketoisocaproate and from endogenous fatty acids were additive. With alpha-ketoisocaproate present as soole substrate at 2 mM, leucine production was less than 10% of alpha-ketoisocaproate uptake and only 30% of the acetyl coenzyme A generated was oxidized in the citric acid cycle. Metabolism of alpha-ketoisocaproate was inhibited by fatty acids, alpha-ketoisovalerate, alpha-keto-beta-methylvalerate, and pyruvate. Oxidation of acetyl-CoA generated from alpha-ketoisocaproate was suppressed by oleate and by pyruvate, but was enhanced by lactate. Metabolism between the different branched chain alpha-ketoacids was mutually competitive. When alpha-ketoisocaproate (2 mM) was added in the presence of high pyruvate concentrations (4.4 mM), flux through pyruvate dehydrogenase was decreased, and the proportion of total pyruvate dehydrogenase in the active form (PDHa) also fell. With lactate as substrate, PDHa was only 25% of total activity and was little affected by addition of alpha-ketoisocaproate. These data suggest that enhanced oxidation of acetyl-CoA from alpha-ketoisocaproate by lactate addition is caused by a low activity of pyruvate dehydrogenase combined with increased flux through the citric acid cycle in response to the energy requirements for gluconeogenesis. However, acetyl-CoA generation from pyruvate is apparently insufficiently inhibited by alpha-ketoisocaproate to cause a diversion of acetyl-CoA formed during alpha-ketoisocaproate metabolism from ketone body formation to oxidation in the citric acid cycle. Measurements of the cell contents of CoASH, acetyl-CoA, acid-soluble acyl-CoA, and acid-insoluble fatty acyl-CoA indicated that when the branched chain alpha-ketoacids were added as sole substrate, their oxidation was limited at a step distal to the branched chain alpha-ketoacid dehydrogenase. Acid-soluble acyl-CoA derivatives were depleted after oleate addition in the presence of alpha-ketoisocaproate, suggesting an inhibition of the branched chain alpha-ketoacid dehydrogenase by the elevation of the mitochondrial NADH/NAD+ ratio observed during fatty acid oxidation. This effect was not observed in the presence of oleate and 2-tetradecylglycidic acid.     

Purification and properties of acyl coenzyme A dehydrogenases from bovine liver. Formation of 2-trans,4-cis-decadienoyl coenzyme A

Three straight chain acyl-CoA dehydrogenases were purified to apparent homogeneity from bovine liver using 40-70% (NH4)2SO4 precipitation, gel filtration, DEAE-cellulose column chromatography, and preparative electrophoresis. Separation of the acyl-CoA dehydrogenases by these procedures has been efficiently monitored by two newly developed analytical methods: (i) native staining of acyl-CoA dehydrogenases following separation by electrophoresis in polyacrylamide gels and (ii) determination of general acyl-CoA dehydrogenase by means of a specific substrate, 4-cis-decenoyl-CoA. The three acyl-CoA dehydrogenases were classified into short chain, general, and long chain acyl-CoA dehydrogenases on the basis of their chain length specificities according to the nomenclature proposed by Hall and Kamin (Hall, C. L., and Kamin, H. (1975) J. Biol. Chem. 250, 3470-3486). The enzymes gave single protein bands in polyacrylamide gel electrophoresis under denaturing and nondenaturing conditions, and their subunit and native molecular weights were estimated to be 40,300 and 188,000 for short chain acyl-CoA dehydrogenase, 43,300 and 205,000 for general acyl-CoA dehydrogenase, and 45,200 and 172,000 for long chain acyl-CoA dehydrogenase. Long chain and general acyl-CoA dehydrogenases markedly differed in their substrate specificities toward unsaturated acyl-CoA esters with a double bond at position 4. The former oxidized 4-cis-decenoyl-CoA at a rate of only 2.7% of that obtained with decanoyl-CoA as substrate, while for the latter enzyme 4-cis-decenoyl-CoA was even a slightly better substrate than decanoyl-CoA. 2-trans,4-cis-Decenoyl-CoA was identified as the product of this reaction.     

3-Hydroxyacyl-CoA dehydrogenase and short chain 3-hydroxyacyl-CoA dehydrogenase in human health and disease

3-Hydroxyacyl-CoA dehydrogenase (HAD) functions in mitochondrial fatty acid beta-oxidation by catalyzing the oxidation of straight chain 3-hydroxyacyl-CoAs. HAD has a preference for medium chain substrates, whereas short chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) acts on a wide spectrum of substrates, including steroids, cholic acids, and fatty acids, with a preference for short chain methyl-branched acyl-CoAs. Therefore, HAD should not be referred to as SCHAD. SCHAD is not a member of the HAD family, but instead, belongs to the short chain dehydrogenase/reductase superfamily. Previously reported cases of SCHAD deficiency are due to an inherited HAD deficiency. SCHAD, also known as 17beta-hydroxysteroid dehydrogenase type 10, is important in brain development and aging. Abnormal levels of SCHAD in certain brain regions may contribute to the pathogenesis of some neural disorders. The human SCHAD gene and its protein product, SCHAD, are potential targets for intervention in conditions, such as Alzheimer's disease, Parkinson's disease, and an X-linked mental retardation, that may arise from the impaired degradation of branched chain fatty acid and isoleucine.     

A novel approach to the characterization of substrate specificity in short/branched chain Acyl-CoA dehydrogenase

Rat and human short/branched chain acyl-CoA dehydrogenases exhibit key differences in substrate specificity despite an overall amino acid identity of 85% between them. Rat short/branched chain acyl-CoA dehydrogenases (SBCAD) are more active toward substrates with longer carbon side chains than human SBCAD, whereas the human enzyme utilizes substrates with longer primary carbon chains. The mechanism underlying this difference in substrate specificity was investigated with a novel surface plasmon resonance assay combined with absorbance and circular dichroism spectroscopy, and kinetics analysis of wild type SBCADs and mutants with altered amino acid residues in the substrate binding pocket. Results show that a relatively few amino acid residues are critical for determining the difference in substrate specificity seen between the human and rat enzymes and that alteration of these residues influences different portions of the enzyme mechanism. Molecular modeling of the SBCAD structure suggests that position 104 at the bottom of the substrate binding pocket is important in determining the length of the primary carbon chain that can be accommodated. Conformational changes caused by alteration of residues at positions 105 and 177 directly affect the rate of electron transfer in the dehydrogenation reactions, and are likely transmitted from the bottom of the substrate binding pocket to beta-sheet 3. Differences between the rat and human enzyme at positions 383, 222, and 220 alter substrate specificity without affecting substrate binding. Modeling predicts that these residues combine to determine the distance between the flavin ring of FAD and the catalytic base, without changing the opening of the substrate binding pocket.     

Localization of the human gene for the El alpha subunit of branched chain keto acid dehydrogenase (BCKDHA) to chromosome 19q13.1----q13.2

The gene encoding the El alpha subunit of branched chain keto acid dehydrogenase (BCKDHA) was mapped to human chromosome region 19q13.1----q13.2 using 3H-labeled cDNA hybridized in situ to human chromosomes.     

Sulfides impair short chain fatty acid β-oxidation at acyl-CoA dehydrogenase level in colonocytes: Implications for ulcerative colitis

The disease process of ulcerative colitis (UC) is associated with a block in -oxidation of short chain fatty acid in colonic epithelial cells which can be reproduced by exposure of cells to sulfides. The aim of the current work was to assess the level in the -oxidation pathway at which sulfides might be inhibitory in human colonocytes. Isolated human colonocytes from cases without colitis (n = 12) were exposed to sulfide (1.5 mM) in the presence or absence of exogenous CoA and ATP. Short chain acyl-CoA esters were measured by a high performance liquid chromatographic assay. ¹⁴CO₂ generation was measured from [1-¹⁴C]butyrate and [6-¹⁴C]glucose. ¹⁴CO₂ from butyrate was significantly reduced (p < 0.001) by sulfide. When colonocytes were incubated with hydrogen sulfide in the presence of CoA and ATP, butyryl-CoA concentration was increased (p < 0.01), while crotonyl-CoA (p < 0.01) and acetyl-CoA (p < 0.01) concentrations were decreased. These results show that sulfides inhibit short chain acyl-CoA dehydrogenase. As oxidation of n-butyrate governs the epithelial barrier function of colonocytes the functional activity of short chain acyl-CoA dehydrogenase may be critical in maintaining colonic mucosal integrity. Maintaining the functional activity of dehydrogenases could be an important determinant in the expression of ulcerative colitis.     

Novel fatty acid beta-oxidation enzymes in rat liver mitochondria. I. Purification and properties of very-long-chain acyl-coenzyme A dehydrogenase.

Freeze-thawed rat liver mitochondria were extensively washed with potassium phosphate, pH 7.5, and the residue was extracted with 10 mM potassium phosphate, pH 7.5, 1% (w/v) sodium cholate, 0.5 M KCl. The four beta-oxidation enzyme activities of the washes and the last extract were assayed with substrates of various carbon chain lengths. Our data suggest that the last extract contains a novel acyl-CoA dehydrogenase and long-chain 3-hydroxyacyl-CoA dehydrogenase. A novel acyl-CoA dehydrogenase was purified. The molecular masses of the native enzyme and the subunit were estimated to be 150 and 71 kDa, respectively. One mole of enzyme contained 2 mole of FAD. These properties and immunochemical properties of the enzyme differed from those of three other acyl-CoA dehydrogenases: short-, medium-, and long-chain acyl-CoA dehydrogenases. Carbon chain length specificity of the enzyme differed from that of other acyl-CoA dehydrogenases. The enzyme was active toward CoA esters of long- and very-long-chain fatty acids, but not toward those of medium- and short-chain fatty acids. The specific enzyme activity was greater than 10 times that of long-chain acyl-CoA dehydrogenase when palmitoyl-CoA was used as substrate. We propose the name "very-long-chain acyl-CoA dehydrogenase" for this enzyme.     

Some physiological functions of the L-leucine dehydrogenase in Bacillus subtilis

Mutants of Bacillus subtilis constitutive for L-leucine dehydrogenase synthesis were selected. Using these mutants we could determine two functional roles for the L-leucine dehydrogenase. This enzyme liberates ammonium ions from branched chain amino acids when supplied as the sole nitrogen source. Another function is to synthesize from L-isoleucine, L-leucine, and L-valine the branched chain -keto acids which are precursors of branched chain fatty acid biosynthesis. These results together with the inducibility of the enzyme suggest that the L-leucine dehydrogenase has primarily a catabolic rather than an anabolic function in the metabolism of Bacillus subtilis.     

Comparative studies of Acyl-CoA dehydrogenases for monomethyl branched chain substrates in amino acid metabolism

Short/branched chain acyl-CoA dehydrogenase (SBCAD), isovaleryl-CoA dehydrogenase (IVD), and isobutyryl-CoA dehydrogenase (IBD) are involved in metabolism of isoleucine, leucine, and valine, respectively. These three enzymes all belong to acyl-CoA dehydrogenase (ACD) family, and catalyze the dehydrogenation of monomethyl branched-chain fatty acid (mmBCFA) thioester derivatives. In the present work, the catalytic properties of rat SBCAD, IVD, and IBD, including their substrate specificity, isomerase activity, and enzyme inhibition, were comparatively studied. Our results indicated that SBCAD has its catalytic properties relatively similar to those of straight-chain acyl-CoA dehydrogenases in terms of their isomerase activity and enzyme inhibition, while IVD and IBD are different. IVD has relatively broader substrate specificity than those of the other two enzymes in accommodating various substrate analogs. The present study increased our understanding for the metabolism of monomethyl branched-chain fatty acids (mmBCFAs) and branched-chain amino acids (BCAAs), which should also be useful for selective control of a particular reaction through the design of specific inhibitors.     

Cofactors and metabolites as potential stabilizers of mitochondrial acyl-CoA dehydrogenases

Protein misfolding is a hallmark of a number of metabolic diseases, in which fatty acid oxidation defects are included. The latter result from genetic deficiencies in transport proteins and enzymes of the mitochondrial β-oxidation, and milder disease conditions frequently result from conformational destabilization and decreased enzymatic function of the affected proteins. Small molecules which have the ability to raise the functional levels of the affected protein above a certain disease threshold are thus valuable tools for effective drug design. In this work we have investigated the effect of mitochondrial cofactors and metabolites as potential stabilizers in two β-oxidation acyl-CoA dehydrogenases: short chain acyl-CoA dehydrogenase and the medium chain acyl-CoA dehydrogenase as well as glutaryl-CoA dehydrogenase, which is involved in lysine and tryptophan metabolism. We found that near physiological concentrations (low micromolar) of FAD resulted in a spectacular enhancement of the thermal stabilities of these enzymes and prevented enzymatic activity loss during a 1h incubation at 40°C. A clear effect of the respective substrate, which was additive to that of the FAD effect, was also observed for short- and medium-chain acyl-CoA dehydrogenase but not for glutaryl-CoA dehydrogenase. In conclusion, riboflavin may be beneficial during feverish crises in patients with short- and medium-chain acyl-CoA dehydrogenase as well as in glutaryl-CoA dehydrogenase deficiencies, and treatment with substrate analogs to butyryl- and octanoyl-CoAs could theoretically enhance enzyme activity for some enzyme proteins with inherited folding difficulties.     

Assay of Acyl-CoA Dehydrogenase Activity in Frozen Muscle Biopsies: Application to Medium-Chain Acyl-CoA Dehydrogenase Deficiency

The acyl-CoA dehydrogenases (ACDs) are mitochondrial enzymes that dehydrogenate acyl-coenzyme A esters of different chain lengths. Inherited deficiencies of these dehydrogenases are commonly associated with muscle weakness and lipid storage. Numerous assays including spectrophotometric, fluorometric, chemical, and radiochemical procedures have been used, but there is need for a rapid, reproducible assay for the different acyl-CoA dehydrogenases in small frozen samples of human muscle biopsies. We describe a comparative study of dye-linked spectrophotometric assays of the long, medium, and short chain acyl-CoA dehydrogenases in frozen rat and human muscle samples. An optimal procedure is described confirming the value of glass-glass homogenization and assay of a 600g supernatant. Higher activities for all acyl-CoA dehydrogenases, citrate synthase, and cytochrome c oxidase were obtained in rat in contrast to human. The substrate-linked dye reduction method was found superior to the ferricenium or electron transfer flavoprotein acceptor systems. Application of the phenazine ethosulfate-DCPIP-linked method to medium-chain acyl-CoA dehydrogenase (MCAD) was studied in detail and the effect of immunoprecipitation of MCAD allowed for the determination of substrate specificity and the degree of crossover between long-, medium-, and short-chain ACD activity following immunoprecipitation. Finally, a comparison of the specificity and validity of the assay in a patient with MCAD deficiency was performed.     

Production of pikromycin using branched chain amino acid catabolism in <Emphasis Type="Italic">Streptomyces venezuelae</Emphasis> ATCC 15439

Branched chain amino acids (BCAA) are catabolized into various acyl-CoA compounds, which are key precursors used in polyketide productions. Because of that, BCAA catabolism needs fine tuning of flux balances for enhancing the production of polyketide antibiotics. To enhance BCAA catabolism for pikromycin production in Streptomyces venezuelae ATCC 15439, three key enzymes of BCAA catabolism, 3-ketoacyl acyl carrier protein synthase III, acyl-CoA dehydrogenase, and branched chain α-keto acid dehydrogenase (BCDH) were manipulated. BCDH overexpression in the wild type strain resulted in 1.3 fold increase in pikromycin production compared to that of WT, resulting in total 25 mg/L of pikromycin. To further increase pikromycin production, methylmalonyl-CoA mutase linked to succinyl-CoA production was overexpressed along with BCDH. Overexpression of the two enzymes resulted in the highest titer of total macrolide production of 43 mg/L, which was about 2.2 fold increase compared to that of the WT. However, it accumulated and produced dehydroxylated forms of pikromycin and methymycin, including their derivatives as well. It indicated that activities of pikC, P450 monooxygenase, newly became a bottleneck in pikromycin synthesis.     

Differential protein expression during growth on linear versus branched alkanes in the obligate marine hydrocarbon-degrading bacterium Alcanivorax borkumensis SK2T

Alcanivorax borkumensis SK2^T is an important obligate hydrocarbonoclastic bacterium (OHCB) that can dominate microbial communities following marine oil spills. It possesses the ability to degrade branched alkanes which provides it a competitive advantage over many other marine alkane degraders that can only degrade linear alkanes. We used LC–MS/MS shotgun proteomics to identify proteins involved in aerobic alkane degradation during growth on linear (n-C₁₄) or branched (pristane) alkanes. During growth on n-C₁₄, A. borkumensis expressed a complete pathway for the terminal oxidation of n-alkanes to their corresponding acyl-CoA derivatives including AlkB and AlmA, two CYP153 cytochrome P450s, an alcohol dehydrogenase and an aldehyde dehydrogenase. In contrast, during growth on pristane, an alternative alkane degradation pathway was expressed including a different cytochrome P450, an alcohol oxidase and an alcohol dehydrogenase. A. borkumensis also expressed a different set of enzymes for β-oxidation of the resultant fatty acids depending on the growth substrate utilized. This study significantly enhances our understanding of the fundamental physiology of A. borkumensis SK2^T by identifying the key enzymes expressed and involved in terminal oxidation of both linear and branched alkanes. It has also highlights the differential expression of sets of β-oxidation proteins to overcome steric hinderance from branched substrates.     

Molecular cloning and nucleotide sequence of cDNA encoding the entire precursor of rat liver medium chain acyl coenzyme A dehydrogenase

cDNA encoding the precursor of rat liver medium chain acyl-CoA dehydrogenase (EC 1.3.99.3) was cloned and sequenced. The longest cDNA insert isolated was 1866 bases in length. This cDNA encodes the entire protein of 421-amino acids including a 25-amino acid leader peptide and a 396-amino acid mature polypeptide. The identity of the medium chain acyl-CoA dehydrogenase clone was confirmed by matching the amino acid sequence predicted from the cDNA to the NH2-terminal and nine internal tryptic peptide sequences derived from pure rat liver medium chain acyl-CoA dehydrogenase. The calculated molecular masses of the precursor medium chain acyl-CoA dehydrogenase, the mature medium chain acyl-CoA dehydrogenase, and the leader peptide are 46,600, 43,700, and 2,900 daltons, respectively. The leader peptide contains five basic amino acids and only one acidic amino acid; thus, it is positively charged, overall. Cysteine residues are unevenly distributed in the mature portion of the protein; five of six are found within the NH2-terminal half of the polypeptide. Comparison of medium chain acyl-CoA dehydrogenase sequence to other flavoproteins and enzymes which act on coenzyme A ester substrates did not lead to unambiguous identification of a possible FAD-binding site nor a coenzyme A-binding domain. The sequencing of other homologous acyl-CoA dehydrogenases will be informative in this regard.     

Murine branched chain α-ketoacid dehydrogenase kinase; cDNA cloning,tissue distribution,and temporal expression during embryonic development

These studies were designed to demonstrate the structural and functional similarity of murine branched chain α-ketoacid dehydrogenase and its regulation by the complex-specific kinase. Nucleotide sequence and deduced amino acid sequence for the kinase cDNA demonstrate a highly conserved coding sequence between mouse and human. Tissue-specific expression in adult mice parallels that reported in other mammals. Kinase expression in female liver is influenced by circadian rhythm. Of special interest is the fluctuating expression of this kinase during embryonic development against the continuing increase in the catalytic subunits of this mitochondrial complex during development. The need for regulation of the branched chain α-ketoacid dehydrogenase complex by kinase expression during embryogenesis is not understood. However, the similarity of murine branched chain α-ketoacid dehydrogenase and its kinase to the human enzyme supports the use of this animal as a model for the human system.     

Aspergillus nidulans contains six possible fatty acyl-CoA synthetases with FaaB being the major synthetase for fatty acid degradation

Aspergillus nidulans can use a variety of fatty acids as sole carbon and energy sources via its peroxisomal and mitochondrial β-oxidation pathways. Prior to channelling the fatty acids into β-oxidation, they need to be activated to their acyl-CoA derivates. Analysis of the genome sequence identified a number of possible fatty acyl-CoA synthetases (FatA, FatB, FatC, FatD, FaaA and FaaB). FaaB was found to be the major long-chain synthetase for fatty acid degradation. FaaB was shown to localise to the peroxisomes, and the corresponding gene was induced in the presence of short and long chain fatty acids. Deletion of the faaB gene leads to a reduced/abolished growth on a variety of fatty acids. However, at least one additional fatty acyl-CoA synthetase with a preference for short chain fatty acids and a potential mitochondrial candidate (AN4659.3) has been identified via genome analysis.