Search

Crystal structures of human long-chain acyl-CoA dehydrogenase (LCAD) and the catalytically inactive Glu291Gln mutant, have been determined. These structures suggest that LCAD harbors functions beyond its historically defined role in mitochondrial β-oxidation of long and medium-chain fatty acids. LCAD is a homotetramer containing one FAD per 43 kDa subunit with Glu291 as the catalytic base. The substrate binding cavity of LCAD reveals key differences which makes it specific for longer and branched chain substrates. The presence of Pro132 near the start of the E helix leads to helix unwinding that, together with adjacent smaller residues, permits binding of bulky substrates such as 3α, 7α, l2α-trihydroxy-5β-cholestan-26-oyl-CoA. This structural element is also utilized by ACAD11, a eucaryotic ACAD of unknown function, as well as bacterial ACADs known to metabolize sterol substrates. Sequence comparison suggests that ACAD10, another ACAD of unknown function, may also share this substrate specificity. These results suggest that LCAD, ACAD10, ACAD11 constitute a distinct class of eucaryotic acyl CoA dehydrogenases., (© 2024. The Author(s).)

Objective: Pediatric familial dilated cardiomyopathy (DCM) is a rare and severe heart disease. The genetics of familial DCM are complex and include over 100 known disease-causing genes, but many causative genes are unknown. We aimed to identify the causative gene for DCM in a consanguineous Saudi Arabian family with affected family members and a history of sudden death., Patients and Methods: Affected (two children) and unaffected (one sibling and the mother) family members were screened by next-generation sequencing (NGS) for 181 candidate DCM genes and underwent metabolic screening. Fifty-seven clinically annotated controls and 46 DCM cases were then tested for the identified mutation. In silico structural and functional analyses including protein modeling, structure prediction and dynamic simulations were performed., Results: A homozygous missense mutation in exon 15 of the acyl-CoA dehydrogenase very long chain gene (ACADVL; chr17:7127303; G>A) was identified in affected subjects that substituted histidine for arginine at codon 450 (p.R450H). The variant was heterozygous in the mother and unaffected sister. The mutation was absent in 57 clinically annotated controls and 48 pediatric DCM cases. The mutation was predicted to cause a significant and deleterious change in the ACADVL protein structure that affected drug binding, stability, and conformation. Metabolic screening confirmed VLCAD deficiency in affected individuals., Conclusions: The ACADVL R450H mutation is an uncommon cause of the DCM phenotype that appears to be autosomal recessive. Targeted NGS is useful for identifying the causative mutation(s) in familial DCM of unknown genetic cause.

Background/objectives: The mitochondrial β-oxidation of fatty acids is a complex catabolic pathway. One of the enzymes of this pathway is the heterooctameric mitochondrial trifunctional protein (MTP), composed of four α- and β-subunits. Mutations in MTP genes (HADHA and HADHB), both located on chromosome 2p23, cause MTP deficiency, a rare autosomal recessive metabolic disorder characterized by decreased activity of MTP. The most common MTP mutation is long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency caused by the c.1528G>C (rs137852769, p.Glu510Gln) substitution in exon 15 of the HADHA gene., Subjects/methods: We analyzed the frequency of genetic variants in the HADHA gene in the adults of Kashubian origin from North Poland and compared this data in other Polish provinces., Results: We found a significantly higher frequency of HDHA c.1528G>C (rs137852769, p.Glu510Gln) carriers among Kashubians (1/57) compared to subjects from other regions of Poland (1/187). We found higher frequency of c.652G>C (rs71441018, pVal218Leu) polymorphism in the HADHA gene within population of Silesia, southern Poland (1/107) compared to other regions., Conclusion: Our study indicate described high frequency of c.1528G>C variant of HADHA gene in Kashubian population, suggesting the founder effect. For the first time we have found high frequency of rs71441018 in the South Poland Silesian population.

Very-long-chain acyl-CoA dehydrogenase deficiency (VLCADD; OMIM#201475) is a rare metabolic disorder of mitochondrial fatty acid oxidation. VLCADD includes three clinical forms that are grouped based on disease severity. Here, we present two unrelated patients suspected of having VLCADD based on a newborn screening test. One patient was diagnosed in the neonatal period and, to date, has not shown any symptoms or signs associated with VLCADD; in contrast, diagnosis was delayed in the other patient after events of hypoketotic hypoglycemia and steatohepatitis. Repeated biochemical analyses and a liver biopsy implied VLCADD, and direct sequencing analysis led to the discovery of three novel mutations, including an identical missense variant (p.Ser207Pro) on ACADVL. Our patients were the first cases of the milder form of VLCADD, and the identical mutation detected might represent a founder mutation in the Korean population and be associated with the milder phenotype of VLCADD., (© 2016 by the Association of Clinical Scientists, Inc.)

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is characterized by impaired mitochondrial β-oxidation of fatty acids. The fatty acid oxidation plays a significant role in energy production especially in skeletal muscle. VLCAD is one of four acyl-CoA dehydrogenases with different-chain length specificity and catalyzes the initial step in mitochondrial β-oxidation of fatty acids. While the clinical phenotypes in neonates and infants are described as severe, adolescent-onset or adult-onset VLCAD deficiency has a more benign course with only skeletal muscle involvement. These myopathic phenotypes are characterized by episodic muscle weakness and rhabdomyolysis triggered by fasting and strenuous exercise. We report a male teenager who manifested repeated episodes of rhabdomyolysis immediately after exertional exercise. Rhabdomyolysis was diagnosed based on the marked elevation of serum creatine kinase and myoglobinuria. Acylcarnitine analysis by tandem mass spectrometry (MS/MS) revealed elevation of serum tetradecenoylcarnitine (C14:1-AC), which represents an abnormal acylcarnitine profile associated with the mitochondrial β-oxidation defect. High performance liquid chromatographic analysis showed decreased production of 2-hexadecenoyl-CoA (C16:1) from palmitoyl-CoA (C16:0), indicating the defect of VLCAD activity. Direct sequencing of the acyl-CoA dehydrogenase, very long-chain gene (ACADVL) that codes VLCAD revealed a heterozygous mutation (c.1242G>C) in exon 12 (E414D), which is a novel mutation in myopathic-type VLCAD deficiency. Because VLCAD functions as a homodimer, we assume that this heterozygous mutation may exhibit dominant-negative effect. This patient remains asymptomatic thereafter by avoiding exertional exercise. The findings of reduction of enzyme activity and clinical features associated with this novel missense mutation of VLCAD are discussed.

SIRT3 and SIRT5 have been shown to regulate mitochondrial fatty acid oxidation but the molecular mechanisms behind the regulation are lacking. Here, we demonstrate that SIRT3 and SIRT5 both target human very long-chain acyl-CoA dehydrogenase (VLCAD), a key fatty acid oxidation enzyme. SIRT3 deacetylates and SIRT5 desuccinylates K299 which serves to stabilize the essential FAD cofactor in the active site. Further, we show that VLCAD binds strongly to cardiolipin and isolated mitochondrial membranes via a domain near the C-terminus containing lysines K482, K492, and K507. Acetylation or succinylation of these residues eliminates binding of VLCAD to cardiolipin. SIRT3 deacetylates K507 while SIRT5 desuccinylates K482, K492, and K507. Sirtuin deacylation of recombinant VLCAD rescues membrane binding. Endogenous VLCAD from SIRT3 and SIRT5 knockout mouse liver shows reduced binding to cardiolipin. Thus, SIRT3 and SIRT5 promote fatty acid oxidation by converging upon VLCAD to promote its activity and membrane localization. Regulation of cardiolipin binding by reversible lysine acylation is a novel mechanism that is predicted to extrapolate to other metabolic proteins that localize to the inner mitochondrial membrane.

Suppressor of cytokine signaling 3 (SOCS3) is a likely mediator of feedback inhibition on the leptin receptor and may cause physiological leptin-resistance, leading to the development of obesity. The aim of this study was to identify potential peptides interacting with purified SOCS3 by using a phage-display human liver cDNA library. We developed a T7 select phage-display system with purified SOCS3 as bait to screen a human liver cDNA library. After 4 rounds of screening and sequencing analysis, we found that phage-presenting peptide RGGVVTSNPLGF show significant binding to SOCS3. The peptide sequence was similar to the sequence of amino acids 644-655 of C-terminal extra-polypeptide of very-long-chain acyl-CoA dehydrogenase (VLCAD), which is 1 of 4 flavoproteins that catalyzing the initial step of the mitochondrial fatty acid β-oxidation, implying a close relationship between SOCS3 and VLCAD. We identified VLCAD as a novel SOCS3 interacting protein both in vitro and vivo, and found that SOCS3 mediates the ubiquitination pathway for proteasomal degradation of VLCAD C-terminal extra-polypeptide via its SOCS-box. Animal experimentation demonstrated that VLCAD is functionally involved in SOCS3 binding and thus, SOCS3 play an important role in the regulation of fatty acid β-oxidation. In conclusion, SOCS3 is an important factor for lipid metabolism and a potential drug-target for treatment of widespread obesity., (Copyright © 2011. Published by Elsevier Inc.)

Long-chain fatty acids are an important source of energy in muscle and heart where the acyl-CoA dehydrogenases (ACADs) participate in consecutive cycles of β-oxidation to generate acetyl-CoA and reducing equivalents for generating energy. However, the role of long-chain fatty acid oxidation in the brain and other tissues that do not rely on fat for energy is poorly understood. Here we characterize two new ACADs, ACAD10 and ACAD11, both with significant expression in human brain. ACAD11 utilizes substrates with primary carbon chain lengths between 20 and 26, with optimal activity towards C22CoA. The combination of ACAD11 with the newly characterized ACAD9 accommodates the full spectrum of long chain fatty acid substrates presented to mitochondrial β-oxidation in human cerebellum. ACAD10 has significant activity towards the branched-chain substrates R and S, 2 methyl-C15-CoA and is highly expressed in fetal but not adult brain. This pattern of expression is similar to that of LCAD, another ACAD previously shown to be involved in long branched chain fatty acid metabolism. Interestingly, the ACADs in human cerebellum were found to have restricted cellular distribution. ACAD9 was most highly expressed in the granular layer, ACAD11 in the white matter, and MCAD in the molecular layer and axons of specific neurons. This compartmentalization of ACADs in the human central nerve system suggests that β-oxidation in cerebellum participates in different functions other than generating energy, for example, the synthesis and/or degradation of unique cellular lipids and catabolism of aromatic amino acids, compounds that are vital to neuronal function., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Acyl-CoA dehydrogenase 9 (ACAD9) is a recently identified member of the acyl-CoA dehydrogenase family. It closely resembles very long-chain acyl-CoA dehydrogenase (VLCAD), involved in mitochondrial beta oxidation of long-chain fatty acids. Contrary to its previously proposed involvement in fatty acid oxidation, we describe a role for ACAD9 in oxidative phosphorylation. ACAD9 binds complex I assembly factors NDUFAF1 and Ecsit and is specifically required for the assembly of complex I. Furthermore, ACAD9 mutations result in complex I deficiency and not in disturbed long-chain fatty acid oxidation. This strongly contrasts with its evolutionary ancestor VLCAD, which we show is not required for complex I assembly and clearly plays a role in fatty acid oxidation. Our results demonstrate that two closely related metabolic enzymes have diverged at the root of the vertebrate lineage to function in two separate mitochondrial metabolic pathways and have clinical implications for the diagnosis of complex I deficiency., (2010 Elsevier Inc. All rights reserved.)

Very-Long-Chain Acyl-CoA Dehydrogenase deficiency (VLCADD) is an autosomal recessive disorder considered as one of the more common ss-oxidation defects, possibly associated with neonatal cardiomyopathy, infantile hepatic coma, or adult-onset myopathy. Numerous gene missense mutations have been described in these VLCADD phenotypes, but only few of them have been structurally and functionally analyzed, and the molecular basis of disease variability is still poorly understood. To address this question, we first analyzed fourteen disease-causing amino acid changes using the recently described crystal structure of VLCAD. The predicted effects varied from the replacement of amino acid residues lining the substrate binding cavity, involved in holoenzyme-FAD interactions or in enzyme dimerisation, predicted to have severe functional consequences, up to amino acid substitutions outside key enzyme domains or lying on near enzyme surface, with predicted milder consequences. These data were combined with functional analysis of residual fatty acid oxidation (FAO) and VLCAD protein levels in patient cells harboring these mutations, before and after pharmacological stimulation by bezafibrate. Mutations identified as detrimental to the protein structure in the 3-D model were generally associated to profound FAO and VLCAD protein deficiencies in the patient cells, however, some mutations affecting FAD binding or monomer-monomer interactions allowed a partial response to bezafibrate. On the other hand, bezafibrate restored near-normal FAO rates in some mutations predicted to have milder consequences on enzyme structure. Overall, combination of structural, biochemical, and pharmacological analysis allowed assessment of the relative severity of individual mutations, with possible applications for disease management and therapeutic approach., (Copyright 2010 Elsevier B.V. All rights reserved.)

Acyl-CoA dehydrogenase [acyl-CoA:(acceptor) 2,3-oxidoreductase; EC 1.3.99.3] catalyzes the first reaction step in mitochondrial fatty-acid beta-oxidation. Here, the very-long-chain acyl-CoA dehydrogenase from Caenorhabditis elegans (cVLCAD) has been cloned and overexpressed in Escherichia coli strain BL21 (DE3). Interestingly, unlike other very-long-chain acyl-CoA dehydrogenases, cVLCAD was found to form a tetramer by size-exclusion chromatography coupled with in-line static light-scattering, refractive-index and ultraviolet measurements. Purified cVLCAD (12 mg ml(-1)) was successfully crystallized by the hanging-drop vapour-diffusion method under conditions containing 100 mM Tris-HCl pH 8.0, 150 mM sodium chloride, 200 mM magnesium formate and 13% PEG 3350. The crystal has a tetragonal form and a complete diffraction data set was collected and processed to 1.8 A resolution. The crystal belonged to space group C2, with unit-cell parameters a = 138.6, b = 116.7, c = 115.3 A, alpha = gamma = 90.0, beta = 124.0 degrees . A self-rotation function indicated the existence of one noncrystallographic twofold axis. A preliminary molecular-replacement solution further confirmed the presence of two molecules in one asymmetric unit, which yields a Matthews coefficient V(M) of 2.76 A(3) Da(-1) and a solvent content of 55%.

Sirtuins are NAD(+)-dependent protein deacetylases. They mediate adaptive responses to a variety of stresses, including calorie restriction and metabolic stress. Sirtuin 3 (SIRT3) is localized in the mitochondrial matrix, where it regulates the acetylation levels of metabolic enzymes, including acetyl coenzyme A synthetase 2 (refs 1, 2). Mice lacking both Sirt3 alleles appear phenotypically normal under basal conditions, but show marked hyperacetylation of several mitochondrial proteins. Here we report that SIRT3 expression is upregulated during fasting in liver and brown adipose tissues. During fasting, livers from mice lacking SIRT3 had higher levels of fatty-acid oxidation intermediate products and triglycerides, associated with decreased levels of fatty-acid oxidation, compared to livers from wild-type mice. Mass spectrometry of mitochondrial proteins shows that long-chain acyl coenzyme A dehydrogenase (LCAD) is hyperacetylated at lysine 42 in the absence of SIRT3. LCAD is deacetylated in wild-type mice under fasted conditions and by SIRT3 in vitro and in vivo; and hyperacetylation of LCAD reduces its enzymatic activity. Mice lacking SIRT3 exhibit hallmarks of fatty-acid oxidation disorders during fasting, including reduced ATP levels and intolerance to cold exposure. These findings identify acetylation as a novel regulatory mechanism for mitochondrial fatty-acid oxidation and demonstrate that SIRT3 modulates mitochondrial intermediary metabolism and fatty-acid use during fasting.

Very-long-chain acyl-CoA dehydrogenase (VLCAD) is a member of the family of acyl-CoA dehydrogenases (ACADs). Unlike the other ACADs, which are soluble homotetramers, VLCAD is a homodimer associated with the mitochondrial membrane. VLCAD also possesses an additional 180 residues in the C terminus that are not present in the other ACADs. We have determined the crystal structure of VLCAD complexed with myristoyl-CoA, obtained by co-crystallization, to 1.91-A resolution. The overall fold of the N-terminal approximately 400 residues of VLCAD is similar to that of the soluble ACADs including medium-chain acyl-CoA dehydrogenase (MCAD). The novel C-terminal domain forms an alpha-helical bundle that is positioned perpendicular to the two N-terminal helical domains. The fatty acyl moiety of the bound substrate/product is deeply imbedded inside the protein; however, the adenosine pyrophosphate portion of the C14-CoA ligand is disordered because of partial hydrolysis of the thioester bond and high mobility of the CoA moiety. The location of Glu-422 with respect to the C2-C3 of the bound ligand and FAD confirms Glu-422 to be the catalytic base. In MCAD, Gln-95 and Glu-99 form the base of the substrate binding cavity. In VLCAD, these residues are glycines (Gly-175 and Gly-178), allowing the binding channel to extend for an additional 12A and permitting substrate acyl chain lengths as long as 24 carbons to bind. VLCAD deficiency is among the more common defects of mitochondrial beta-oxidation and, if left undiagnosed, can be fatal. This structure allows us to gain insight into how a variant VLCAD genotype results in a clinical phenotype.

Very-long-chain acyl-coenzyme A dehydrogenase (VLCAD) deficiency is an inborn mitochondrial fatty-acid beta-oxidation (FAO) defect associated with a broad mutational spectrum, with phenotypes ranging from fatal cardiopathy in infancy to adolescent-onset myopathy, and for which there is no established treatment. Recent data suggest that bezafibrate could improve the FAO capacities in beta-oxidation-deficient cells, by enhancing the residual level of mutant enzyme activity via gene-expression stimulation. Since VLCAD-deficient patients frequently harbor missense mutations with unpredictable effects on enzyme activity, we investigated the response to bezafibrate as a function of genotype in 33 VLCAD-deficient fibroblasts representing 45 different mutations. Treatment with bezafibrate (400 microM for 48 h) resulted in a marked increase in FAO capacities, often leading to restoration of normal values, for 21 genotypes that mainly corresponded to patients with the myopathic phenotype. In contrast, bezafibrate induced no changes in FAO for 11 genotypes corresponding to severe neonatal or infantile phenotypes. This pattern of response was not due to differential inductions of VLCAD messenger RNA, as shown by quantitative real-time polymerase chain reaction, but reflected variable increases in measured VLCAD residual enzyme activity in response to bezafibrate. Genotype cross-analysis allowed the identification of alleles carrying missense mutations, which could account for these different pharmacological profiles and, on this basis, led to the characterization of 9 mild and 11 severe missense mutations. Altogether, the responses to bezafibrate reflected the severity of the metabolic blockage in various genotypes, which appeared to be correlated with the phenotype, thus providing a new approach for analysis of genetic heterogeneity. Finally, this study emphasizes the potential of bezafibrate, a widely prescribed hypolipidemic drug, for the correction of VLCAD deficiency and exemplifies the integration of molecular information in a therapeutic strategy.

The acyl-CoA dehydrogenases are a family of multimeric flavoenzymes that catalyze the alpha,beta -dehydrogenation of acyl-CoA esters in fatty acid beta -oxidation and amino acid catabolism. Genetic defects have been identified in most of the acyl-CoA dehydrogenases in humans. Acyl-CoA dehydrogenase 9 (ACAD9) is a recently identified acyl-CoA dehydrogenase that demonstrates maximum activity with unsaturated long-chain acyl-CoAs. We now report three cases of ACAD9 deficiency. Patient 1 was a 14-year-old, previously healthy boy who died of a Reye-like episode and cerebellar stroke triggered by a mild viral illness and ingestion of aspirin. Patient 2 was a 10-year-old girl who first presented at age 4 mo with recurrent episodes of acute liver dysfunction and hypoglycemia, with otherwise minor illnesses. Patient 3 was a 4.5-year-old girl who died of cardiomyopathy and whose sibling also died of cardiomyopathy at age 21 mo. Mild chronic neurologic dysfunction was reported in all three patients. Defects in ACAD9 mRNA were identified in the first two patients, and all patients manifested marked defects in ACAD9 protein. Despite a significant overlap of substrate specificity, it appears that ACAD9 and very-long-chain acyl-CoA dehydrogenase are unable to compensate for each other in patients with either deficiency. Studies of the tissue distribution and gene regulation of ACAD9 and very-long-chain acyl-CoA dehydrogenase identify the presence of two independently regulated functional pathways for long-chain fat metabolism, indicating that these two enzymes are likely to be involved in different physiological functions.

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is a disorder of fatty acid beta-oxidation that can present at any age with cardiomyopathy, rhabdomyolysis, hepatic dysfunction, and/or nonketotic hypoglycemia. Through the expansion of newborn screening programs an increasing number of individuals with VLCAD deficiency are being identified prior to the onset of symptoms allowing early initiation of therapy. The development of a safe, durable, and effective VLCAD gene delivery system for use at the time of diagnosis could result in a significant improvement in the quality and duration of life for patients with VLCAD deficiency. To this end, we developed a construct containing the human VLCAD cDNA under the control of the strong CMV promoter (pCMV-hVLCAD). A novel rabbit polyclonal anti-VLCAD antibody was prepared using a 24 amino-acid peptide unique to the human VLCAD protein to study human VLCAD expression in immune competent mice. Antibody specificity was demonstrated in Western blots of human VLCAD deficient fibroblasts and in pCMV-hVLCAD transiently transfected VLCAD deficient fibroblasts. Transfected fibroblasts showed correction of the metabolic block as demonstrated by normalization of C14- and C16-acylcarnitine species in cell culture media and restoration of VLCAD activity in cells. Following tail vein injection of pCMV-hVLCAD into mice, we demonstrated expression of hVLCAD in liver. Altogether, these steps are important in the development of a durable gene therapy for VLCAD deficiency.

The de novo synthesis of fatty acids occurs in two distinct cellular compartments. Palmitate (16:0) is synthesized from acetyl-CoA and malonyl-CoA in the cytoplasm by the enzymes acetyl-CoA carboxylase 1 and fatty acid synthase. The synthesis of fatty acids longer than 16 carbons takes place in microsomes and utilizes malonyl-CoA as the carbon source. Each two-carbon addition requires four sequential reactions: condensation, reduction, dehydration, and a final reduction to form the elongated fatty acyl-CoA. The initial condensation reaction is the regulated and rate-controlling step in microsomal fatty acyl elongation. We previously reported the cDNA cloning and characterization of a murine long chain fatty acyl elongase (LCE) . Overexpression of LCE in cells resulted in the enhanced addition of two-carbon units to C12-C16 fatty acids, and evidence was provided that LCE catalyzed the initial condensation reaction of long chain fatty acid elongation. The remaining three enzymes in the elongation reaction have not been identified in mammals. Here, we report the identification and characterization of two mammalian enzymes that catalyze the 3-ketoacyl-CoA and trans-2,3-enoyl-CoA reduction reactions in long and very long chain fatty acid elongation, respectively.

Acyl-CoA dehydrogenases (ACADs) are a family of mitochondrial enzymes catalyzing the initial rate-limiting step in the beta-oxidation of fatty acyl-CoA. The reaction provides main source of energy for human heart and skeletal muscle. Eight human ACADs have been described. Deficiency of these enzymes, especially very long-chain acyl-CoA dehydrogenase (VLCAD), usually leads to severe human organic diseases, such as sudden death in infancy, infantile cardiomyopathy (CM), hypoketotic hypoglycemia, or hepatic dysfunction. By large-scale random sequencing, we identified a novel homolog of ACADs from human dendritic cell (DC) cDNA library. It contains an open reading frame (ORF) of 1866bp, which encodes a 621 amino acid protein. It shares approximately 47% amino acid identity and 65% similarity with human VLCAD. So, the novel molecule is named as acyl-CoA dehydrogenase-9 (ACAD-9), the ninth member of ACADs. The new gene consists of 18 exons and 17 introns, and is mapped to chromosome 3q26. It contains the two signatures shared by all members of the ACADs. ACAD-9 mRNA is ubiquitously expressed in most normal human tissues and cancer cell lines with high level of expression in heart, skeletal muscles, brain, kidney, and liver. Enzymatic assay proved that the recombinant ACAD-9 protein has the dehydrogenase activity on palmitoyl-coenzyme A (C16:0) and stearoyl-coenzyme A (C18:0). Our results indicate that ACAD-9 is a novel member of ACADs.

Salmonella enterica serovar Typhimurium (S. typhimurium) is an enteric pathogen that causes significant morbidity in humans and other mammals. During their life cycle, salmonellae must survive frequent exposures to a variety of environmental stresses, e.g. carbon-source (C) starvation. The starvation-stress response (SSR) of S. typhimurium encompasses the genetic and physiological realignments that occur when an essential nutrient becomes limiting for bacterial growth. The function of the SSR is to produce a cell capable of surviving long-term starvation. This paper reports that three C-starvation-inducible lac fusions from an S. typhimurium C-starvation-inducible lac fusion library are all within a gene identified as fadF, which encodes an acyl-CoA dehydrogenase (ACDH) specific for medium-/long-chain fatty acids. This identification is supported by several findings: (a) significant homology at the amino acid sequence level with the ACDH enzymes from other bacteria and eukaryotes, (b) undetectable beta-oxidation levels in fadF insertion mutants, (c) inability of fad insertion mutants to grow on oleate or decanoate as a sole C-source, and (d) inducibility of fadF::lac fusions by the long-chain fatty acid oleate. In addition, the results indicate that the C-starvation-induction of fadF is under negative control by the FadR global regulator and positive control by the cAMP:cAMP receptor protein complex and ppGpp. It is also shown that the fadF locus is important for C-starvation-survival in S. typhimurium. Furthermore, the results demonstrate that fadF is induced within cultured Madin-Darby canine kidney (MDCK) epithelial cells, suggesting that signals for its induction (C-starvation and/or long-chain fatty acids) may be present in the intracellular environment encountered by S. typhimurium. However, fadF insertion mutations did not have an overt effect on mouse virulence.

Very-long-chain acyl-coenzyme A dehydrogenase (VLCAD) is one of four enzymes which catalyze the initial step of the mitochondrial beta-oxidation with different but overlapping substrate-chain-length specificities. A450P, a variant of VLCAD identified in a patient with VLCAD deficiency, showed abnormal substrate-chain-length specificity. Based on this mutation, we studied the relationship between the structure and substrate-chain-length specificity of VLCAD. When VLCAD was treated with trypsin, a homodimer protein of a 48-kDa polypeptide deprived of both the amino-terminal 22 amino acids and the carboxyl-terminal 145 amino acids of VLCAD was obtained. Six Ala450 variants and tryptic-VLCAD exhibited similar substrate specificities. Effects of long-chain acyl-CoA on the tryptic cleavage and changes in the catalytic properties by deprivation of the carboxyl-terminal region suggest that this region interacts with the fatty acyl moiety of long-chain acyl-CoA. Thus, both Ala450 and the carboxyl-terminal region, which are not shared by other acyl-CoA dehydrogenases, are likely to be the determinating factors in the substrate-chain-length specificity of VLCAD.

Long-chain acyl-CoA dehydrogenase (LCAD) is one of four enzymes involved in the initial step of mitochondrial beta-oxidation of straight-chain fatty acids. It is a member of the acyl-CoA dehydrogenase (Acad or ACAD) gene family of enzymes, which also includes very-long-chain (VLCAD), medium-chain (MCAD), and short-chain (SCAD) acyl-CoA dehydrogenases. These enzymes all have similar activity but differ only in the chain length specificity for their substrate. Mitochondrial beta-oxidation provides an important source of energy especially during times of fasting. In order to understand the role of LCAD in this pathway, we have cloned and characterized the entire mouse (Mus musculus) gene encoding LCAD (Acadl). Acadl is a single-copy, nuclear encoded gene approximately 35 kb in size. We have sequenced the entire coding region, all intron/exon boundaries, 1.7 kb of its 5' regulatory region, and mapped the transcription start site. The gene contains 11 coding exons ranging in size from 67 bp to 275 bp, interrupted by 10 introns ranging in size from 1.0 kb to 6.6 kb in size. The Acadl 5' regulatory region, like other members of the Acad family, lacks a TATA or CAAT box and is GC rich. This region does contain multiple, putative cis-acting DNA elements recognized by either SP1 or members of the steroid-thyroid family of nuclear receptors, which has been shown with other members of the ACAD gene family to be important in regulated expression. The characterization of the mouse Acadl gene will allow further study of LCAD in an in vivo model, and how its expression may be coordinated with other members of the Acad gene family.

Very long-chain acyl-CoA dehydrogenase (VLCAD) is one of four flavoproteins which catalyze the initial step of the mitochondrial beta-oxidation spiral. By sequence comparison with other acyl-CoA dehydrogenases, Glu-422 of VLCAD has been presumed to be the catalytic residue that abstracts the alpha-proton in the alphabeta-dehydrogenation reaction. Replacing Glu-422 with glutamine (E422Q) caused a loss of enzyme activity by preventing the formation of a charge transfer complex between VLCAD and palmitoyl-CoA. This result provides further evidence for Glu-422 being part of the active site of VLCAD. F418L is a disease-causing mutation in human VLCAD deficiency. Unlike wild-type VLCAD, F418L and F418V contained no bound FAD when expressed at extremely high levels in the baculovirus expression system. Although F418T and F418Y bound FAD at a level similar to that of wild-type VLCAD, both showed reduced Vmax values toward palmitoyl-CoA, most likely due to a decrease in the rate of enzyme-bound FAD reduction. These data suggest that Phe-418 is involved in the binding and subsequent reduction of FAD. FAD-deficient VLCADs (F418L, F418V, and apo-VLCAD) showed increased sensitivity to trypsinization. Loss of FAD may change the folding of VLCAD subunit.

Isovaleryl-CoA dehydrogenase (IVD) belongs to an important flavoprotein family of acyl-CoA dehydrogenases that catalyze the alpha,beta-dehydrogenation of their various thioester substrates. Although enzymes from this family share similar sequences, catalytic mechanisms, and structural properties, the position of the catalytic base in the primary sequence is not conserved. E376 has been confirmed to be the catalytic base in medium-chain (MCAD) and short-chain acyl-CoA dehydrogenases and is conserved in all members of the acyl-CoA dehydrogenase family except for IVD and long-chain acyl-CoA dehydrogenase. To understand this dichotomy and to gain a better understanding of the factors important in determining substrate specificity in this enzyme family, the three-dimensional structure of human IVD has been determined. Human IVD expressed in Escherichia coli crystallizes in the orthorhombic space group P212121 with unit cell parameters a = 94.0 A, b = 97.7 A, and c = 181.7 A. The structure of IVD was solved at 2.6 A resolution by the molecular replacement method and was refined to an R-factor of 20.7% with an Rfree of 28.8%. The overall polypeptide fold of IVD is similar to that of other members of this family for which structural data are available. The tightly bound ligand found in the active site of the structure of IVD is consistent with that of CoA persulfide. The identity of the catalytic base was confirmed to be E254, in agreement with previous molecular modeling and mutagenesis studies. The location of the catalytic residue together with a glycine at position 374, which is a tyrosine in all other members of the acyl-CoA dehydrogenase family, is important for conferring branched-chain substrate specificity to IVD.

The acyl-CoA dehydrogenases are a family of flavoenzymes with similar structure and function involved in the metabolism of fatty acids and branched chain amino acids. The degree of overlap in substrate specificity is narrow among these enzymes. The position of the catalytic glutamate, identified as Glu376 in porcine medium chain acyl-CoA dehydrogenase (MCAD), Glu254 in human isovaleryl-CoA dehydrogenase (IVD), and Glu261 in human long chain acyl-CoA dehydrogenase (LCAD), has been suggested to affect substrate chain length specificity. In this study, in vitro site-directed mutagenesis was used to investigate the effect of changing the position of the catalytic carboxylate on substrate specificity in short chain acyl-CoA dehydrogenase (SCAD). Glu368, the hypothetical active site catalytic residue of rat SCAD, was replaced with Asp, Gly, Gln, Arg, and Lys and the wild type and mutant SCADs were produced in Escherichia coli and purified. The recombinant wild type SCAD kcat/K(m) values for butyryl-hexanoyl-, and octanoyl-CoA were 220, 22, and 3.2 microM-1 min-1, respectively, while the Glu368Asp mutant gave kcat/K(m) of 81, 12, and 1.4 microM-1 min-1, respectively, for the same substrates. None of the other mutants exhibited enzyme activity. A Glu368Gly/Gly247Glu double mutant enzyme, which places the catalytic residue at a position homologous to that of LCAD, was also synthesized and purified. It showed kcat/K(m) of 9.3, 2.8, and 1.5 microM-1 min-1 with butyryl-, hexanoyl-, and octanoyl-CoA used as substrates, respectively. These results confirm the identity of Glu368 as the catalytic residue of rat SCAD and suggest that alteration of the position of the catalytic carboxylate can modify substrate specificity.

Medium- and short-chain acyl-CoA oxidases were identified in and subsequently purified from dark-grown maize plantlets. The oxidase showing preference for medium-chain fatty acyl-CoAs (C10-C14) was purified to homogeneity. The oxidase showing preference for short-chain fatty acyl-CoAs (C4-C8) was purified over 150-fold. Various catalytic properties confirmed these enzymes to be true acyl-CoA oxidases. They produced trans-2-enoyl-CoA and H2O2 from the saturated acyl-CoA, as verified by various independent assay techniques. They also exhibited FAD-dependent activity; i.e. removal of loosely bound FAD by gel filtration markedly reduced activity, which could be restored upon re-addition of FAD. They showed apparent Km values between 2 and 10 microM for the acyl-CoA substrate giving maximal activity, no activity with the corresponding free fatty acid, high pH optima (8.3-8.6) and a peroxisomal subcellular location. The medium-chain acyl-CoA oxidase was determined to be a monomeric protein with a molecular mass of 62 kDa. The short-chain acyl-CoA oxidase was shown to have a native molecular mass of 60 kDa, but exhibited a labile multimeric structure, as indicated by the elution of multiple peaks of activity during several chromatographic steps, and ultimately by the purification of a subunit of molecular mass 15 kDa. The medium- and short-chain acyl-CoA oxidases were demonstrated to be distinct from the maize equivalent of the cucumber glyoxysomal long-chain acyl-CoA oxidase previously purified and characterized [Kirsch, Loffler and Kindl (1986) J. Biol. Chem. 261, 8570-8575]. The maize long-chain acyl-CoA oxidase was partially purified to permit determination of its substrate specificity; it showed activity with a broad range of acyl-CoAs of chain length greater than C8, and maximal activity with C16. The implications of the existence of multiple acyl-CoA oxidases in the regulation of plant peroxisomal beta-oxidation are discussed.

The catalytically essential glutamate residue that initiates catalysis by abstracting the substrate alpha-hydrogen as H+ is located at position 376 (mature MCADH numbering) on loop JK in medium chain acyl-CoA dehydrogenase (MCADH). In long chain acyl-CoA dehydrogenase (LCADH) and isovaleryl-CoA dehydrogenase (IVDH), the corresponding Glu carrying out the same function is placed at position 255 on the adjacent helix G. These glutamates thus act on substrate approaching from two opposite regions at the active center. We have implemented the topology of LCADH in MCADH by carrying out the two mutations Glu376Gly and Thr255Glu. The resulting chimeric enzyme, "medium-/long" chain acyl-CoA dehydrogenase (MLCADH) has approximately 20% of the activity of MCADH and approximately 25% that of LCADH with its best substrates octanoyl-CoA and dodecanoyl-CoA, respectively. MLCADH exhibits an enhanced rate of reoxidation with oxygen, however, with a much narrower substrate chain length specificity that peaks with dodecanoyl-CoA. This is the same maximum as that of LCADH and is thus significantly shifted from that of native MCADH (hexanoyl/octanoyl-CoA). The putative, common ancestor of LCADH and IVDH has two Glu residues, one each at positions 255 and 376. The corresponding MCADH mutant, Thr255Glu (glu/glu-MCADH), is as active as MCADH with octanoyl-CoA; its activity/chain length profile is, however, much narrower. The topology of the Glu as H+ abstracting base seems an important factor in determining chain length specificity and reactivity in acyl-CoA dehydrogenases. The mechanisms underlying these effects are discussed in view of the three-dimensional structure of MLCADH, which is presented in the accompanying paper [Lee et al. (1996) Biochemistry 35, 12412-12420].

Crystal structures of the wild type human medium-chain acyl-CoA dehydrogenase (MCADH) and a double mutant in which its active center base-arrangement has been altered to that of long chain acyl-CoA dehydrogenase (LCADH), Glu376Gly/Thr255Glu, have been determined by X-ray crystallography at 2.75 and 2.4 A resolution, respectively. The catalytic base responsible for the alpha-proton abstraction from the thioester substrate is Glu376 in MCADH, while that in LCADH is Glu255 (MCADH numbering), located over 100 residues away in its primary amino acid sequence. The structures of the mutant complexed with C8-, C12, and C14-CoA have also been determined. The human enzyme structure is essentially the same as that of the pig enzyme. The structure of the mutant is unchanged upon ligand binding except for the conformations of a few side chains in the active site cavity. The substrate with chain length longer than C12 binds to the enzyme in multiple conformations at its omega-end. Glu255 has two conformations, "active" and "resting" forms, with the latter apparently stabilized by forming a hydrogen bond with Glu99. Both the direction in which Glu255 approaches the C alpha atom of the substrate and the distance between the Glu255 carboxylate and the C alpha atom are different from those of Glu376; these factors are responsible for the intrinsic differences in the kinetic properties as well as the substrate specificity. Solvent accessible space at the "midsection" of the active site cavity, where the C alpha-C beta bond of the thioester substrate and the isoalloxazine ring of the FAD are located, is larger in the mutant than in the wild type enzyme, implying greater O2 accessibility in the mutant which might account for the higher oxygen reactivity.

We have shown previously that acetoacetyl-CoA bound to medium-chain acyl-CoA dehydrogenase from pig kidney is transformed into an enolate form, O = C(3)-C(2)H = C(1)-O-, and that the interaction between the C(4a) = N(5) moiety of flavin and the O = C(3)-C(2)H = C(1)-O- moiety of acetoacetyl-CoA is important for the charge-transfer interaction [Nishina, Y. et al. (1992) J. Biochem. 111, 699-706]. In this study, we examined four kinds of acyl-CoA dehydrogenases [short-chain acyl-CoA (SCAD), medium-chain acyl-CoA (MCAD), long-chain acyl-CoA (LCAD), and isovaleryl-CoA (IVD) dehydrogenases] from bovine liver. The Raman spectra of non-labeled and isotopically labeled acetoacetyl-CoA in keto-form revealed that the 1,716-cm-1 and 1,650-cm-1 bands were derived from the C(3) = O and the C(1) = O stretching mode, respectively. In the charge-transfer complexes of acetoacetyl-CoA with the four kinds of dehydrogenases, the resonance Raman (RR) bands corresponding to the C(3) = O and the C(1) = O of acetoacetyl-CoA were observed at around 1,643-1,622 and 1,506-1,476 cm-1, respectively, indicating that acetoacetyl-CoA was transformed into the enolate form as the result of the complexation with the enzymes. Further, in RR spectra with excitation at 632.8 nm, within the charge-transfer band of the complexes of acetoacetyl-CoA with the four acyl-CoA dehydrogenases, both bands associated with the C(4a) = N(5) moiety of oxidized flavin and the O = C(3)-C(2)H = C(1)-O- moiety of acetoacetyl-CoA were enhanced, but the benzene portion of oxidized flavin was not. These results indicate that the substrate activating mechanism is common to all four kinds of dehydrogenases, i.e., the interaction between the C(1) = O of acetoacetyl-CoA and the positively polarized atoms of the enzymes located in close proximity to the oxygen atom of C(1) = O is important, and the C(4a) = N(5) moiety of flavin participates in the interaction. Some kinds of 3-ketoacyl-CoAs were tested instead of acetoacetyl-CoA and essentially similar results were obtained. The positions of the bands derived from the C(1)-O- moiety of 3-ketoacyl-CoAs were different by ca. 30 cm-1 in two groups, i.e., ca. 1,475 cm-1 for SCAD and MCAD and ca. 1,505 cm-1 for LCAD and IVD, that is, RR spectra can classify the four dehydrogenases into two groups.

We have used molecular modeling and site-directed mutagenesis to identify the catalytic residues of human long chain acyl-CoA dehydrogenase. Among the acyl-CoA dehydrogenases, a family of flavoenzymes involved in beta-oxidation of fatty acids, only the three-dimensional structure of the medium chain fatty acid specific enzyme from pig liver has been determined (Kim, J.-J.P., Wang, M., & Paschke, R. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7523-7527). Despite the overall sequence homology, the catalytic residue (E376) of medium chain acyl-CoA dehydrogenase is not conserved in isovaleryl- and long chain acyl-CoA dehydrogenases. A molecular model of human long chain acyl-CoA dehydrogenase was derived using atomic coordinates determined by X-ray diffraction studies of the pig medium chain specific enzyme, interactive graphics, and molecular mechanics calculations. The model suggests that E261 functions as the catalytic base in the long-chain dehydrogenase. An altered dehydrogenase in which E261 was replaced by a glutamine was constructed, expressed, purified, and characterized. The mutant enzyme exhibited less than 0.02% of the wild-type activity. These data strongly suggest that E261 is the base that abstracts the alpha-proton of the acyl-CoA substrate in the catalytic pathway of this dehydrogenase.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is a common autosomal recessive disorder of mitochondrial fatty acid oxidation characterized by episodes of hypoketotic hypoglycemia usually beginning in the first 2 y of life. We previously showed, in pulse labeling experiments, that the biosynthesis and immediate posttranslational processing of MCAD are normal in fibroblasts from patients with MCAD deficiency. Most patients studied to date are homozygous for a point mutation (A985-G) that results in the substitution of glutamate for lysine ar residue 304 of the mature MCAD subunit. We performed immunoblot analysis of fibroblast MCAD from a total of 34 patients with MCAD deficiency, including 31 homozygous for the A985-G mutation, using a rabbit anti-rat MCAD antibody that cross-reacted specifically with human MCAD, but not with the related enzymes, short-chain and long-chain acyl-CoA dehydrogenases. All patients with the A985-G mutation lacked detectable MCAD. Pulse-chase labeling of MCAD-deficient fibroblasts with 35S-methionine demonstrated that this variant MCAD was unstable compared to controls. Taken together, these data suggest that this mutation affects the stability of MCAD protein within the mitochondrial matrix.