Molecular cloning of cDNA encoding mitochondrial very-long-chain acyl-CoA dehydrogenase from bovine heart. (1/168)

AIM: To clone the cDNA encoding an isoenzyme of mitochondrial very-long-chain acyl-CoA dehydrogenase (VLCAD) from bovine heart lambda gt11 and lambda gt10 cDNA libraries. METHODS: The clone was isolated with immunoscreening technique and validated by (1) the microsequences of the N-terminus and three internal proteolytic fragments from the purified enzyme; (2) identification of the acyl-CoA dehydrogenase (AD) signature sequence; and (3) high homology of the deduced peptide sequences, as expected, with those of rat liver mitochondrial VLCAD. RESULTS: The cDNA (2203 bp) corresponds to a approximately 2.4-kb mRNA band from the same tissue source revealed by a Northern blotting. The deduced peptide sequence of 655 amino acids (70,537 Da) is composed of a 40-amino acid mitochondrial leader peptide moiety (4,346 Da) and a 615-amino acid peptide as a mature protein (66,191 Da). A comparison of the peptide sequences in the AD family shows the major diversity in their signal sequences, suggesting a structural basis for their different mitochondrial locations. The catalytic sites are all highly conserved among VLCAD. Ser-251 analogous to and Cys-215 diversified to other family members. A pseudo-consensus sequence of leucine zipper was found in the C-terminal region from Leu-568 to Leu-589, implying a mechanism whereby the dimer of this protein is formed by zipping these leucine residues from the alpha-helixes of 2 monomers. CONCLUSION: The isolated cDNA clone encodes an isoenzyme of mitochondrial VLCAD in bovine heart.  (+info)

Molecular heterogeneity in very-long-chain acyl-CoA dehydrogenase deficiency causing pediatric cardiomyopathy and sudden death. (2/168)

BACKGROUND: Genetic defects are being increasingly recognized in the etiology of primary cardiomyopathy (CM). Very-long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes the first step in the beta-oxidation spiral of fatty acid metabolism, the crucial pathway for cardiac energy production. METHODS AND RESULTS: We studied 37 patients with CM, nonketotic hypoglycemia and hepatic dysfunction, skeletal myopathy, or sudden death in infancy with hepatic steatosis, features suggestive of fatty acid oxidation disorders. Single-stranded conformational variance was used to screen genomic DNA. DNA sequencing and mutational analysis revealed 21 different mutations on the VLCAD gene in 18 patients. Of the mutations, 80% were associated with CM. Severe CM in infancy was recognized in most patients (67%) at presentation. Hepatic dysfunction was common (33%). RNA blot analysis and VLCAD enzyme assays showed a severe reduction in VLCAD mRNA in patients with frame-shift or splice-site mutations and absent or severe reduction in enzyme activity in all. CONCLUSIONS: Infantile CM is the most common clinical phenotype of VLCAD deficiency. Mutations in the human VLCAD gene are heterogeneous. Although mortality at presentation is high, both the metabolic disorder and cardiomyopathy are reversible.  (+info)

Oxidation of medium-chain acyl-CoA esters by extracts of Aspergillus niger: enzymology and characterization of intermediates by HPLC. (3/168)

The activities of beta-oxidation enzymes were measured in extracts of glucose- and triolein-grown cells of Aspergillus niger. Growth on triolein stimulated increased enzyme activity, especially for acyl-CoA dehydrogenase. No acyl-CoA oxidase activity was detected. HPLC analysis after incubation of triolein-grown cell extracts with decanoyl-CoA showed that beta-oxidation was limited to one cycle. Octanoyl-CoA accumulated as the decanoyl-CoA was oxidized. Beta-oxidation enzymes in isolated mitochondrial fractions were also studied. The results are discussed in the context of methyl ketone production by fungi.  (+info)

Outcome of medium chain acyl-CoA dehydrogenase deficiency after diagnosis. (4/168)

BACKGROUND: Medium chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common inborn error of fatty acid metabolism. Undiagnosed, it has a mortality rate of 20-25%. Neonatal screening for the disorder is now possible but it is not known whether this would alter the prognosis. OBJECTIVE: To investigate the outcome of MCAD deficiency after the diagnosis has been established. METHOD: All patients with a proved diagnosis of MCAD deficiency attending one centre in a four year period were reviewed. RESULTS: Forty one patients were identified. Follow up was for a median of 6.7 years (range, 9 months to 14 years). Nearly half of the patients were admitted to hospital with symptoms characteristic of MCAD deficiency before the correct diagnosis was made. After diagnosis, two patients were admitted to hospital with severe encephalopathy but there were no additional deaths or appreciable morbidity. There was a high incidence (about one fifth) of previous sibling deaths among the cohort. CONCLUSIONS: Undiagnosed, MCAD deficiency results in considerable mortality and morbidity. However, current management improves outcome, supporting the view that the disorder should be included in newborn screening programmes.  (+info)

A novel acyl-CoA oxidase that can oxidize short-chain acyl-CoA in plant peroxisomes. (5/168)

Short-chain acyl-CoA oxidases are beta-oxidation enzymes that are active on short-chain acyl-CoAs and that appear to be present in higher plant peroxisomes and absent in mammalian peroxisomes. Therefore, plant peroxisomes are capable of performing complete beta-oxidation of acyl-CoA chains, whereas mammalian peroxisomes can perform beta-oxidation of only those acyl-CoA chains that are larger than octanoyl-CoA (C8). In this report, we have shown that a novel acyl-CoA oxidase can oxidize short-chain acyl-CoA in plant peroxisomes. A peroxisomal short-chain acyl-CoA oxidase from Arabidopsis was purified following the expression of the Arabidopsis cDNA in a baculovirus expression system. The purified enzyme was active on butyryl-CoA (C4), hexanoyl-CoA (C6), and octanoyl-CoA (C8). Cell fractionation and immunocytochemical analysis revealed that the short-chain acyl-CoA oxidase is localized in peroxisomes. The expression pattern of the short-chain acyl-CoA oxidase was similar to that of peroxisomal 3-ketoacyl-CoA thiolase, a marker enzyme of fatty acid beta-oxidation, during post-germinative growth. Although the molecular structure and amino acid sequence of the enzyme are similar to those of mammalian mitochondrial acyl-CoA dehydrogenase, the purified enzyme has no activity as acyl-CoA dehydrogenase. These results indicate that the short-chain acyl-CoA oxidases function in fatty acid beta-oxidation in plant peroxisomes, and that by the cooperative action of long- and short-chain acyl-CoA oxidases, plant peroxisomes are capable of performing the complete beta-oxidation of acyl-CoA.  (+info)

Evaluating newborn screening programmes based on dried blood spots: future challenges. (6/168)

A UK national programme to screen all newborn infants for phenylketonuria was introduced in 1969, followed in 1981 by a similar programme for congenital hypothyroidism. Decisions to start these national programmes were informed by evidence from observational studies rather than randomised controlled trials. Subsequently, outcome for affected children has been assessed through national disease registers, from which inferences about the effectiveness of screening have been made. Both programmes are based on a single blood specimen, collected from each infant at the end of the first week of life, and stored as dried spots on a filter paper or 'Guthrie' card. This infrastructure has made it relatively easy for routine screening for other conditions to be introduced at a district or regional level, resulting in inconsistent policies and inequitable access to effective screening services. This variation in screening practices reflects uncertainty and the lack of a national framework to guide the introduction and evaluation of new screening initiatives, rather than geographical variations in disease prevalence or severity. More recently, developments in tandem mass spectrometry have made it technically possible to screen for several inborn errors of metabolism in a single analytical step. However, for each of these conditions, evidence is required that the benefits of screening outweigh the harms. How should that evidence be obtained? Ideally policy decisions about new screening initiatives should be informed by evidence from randomised controlled trials but for most of the conditions for which newborn screening is proposed, large trials would be needed. Prioritising which conditions should be formally evaluated, and developing a framework to support their evaluation, poses an important challenge to the public health, clinical and scientific community. In this chapter, issues underlying the evaluation of newborn screening programmes will be discussed in relation to medium chain acyl CoA dehydrogenase deficiency, a recessively inherited disorder of fatty acid oxidation.  (+info)

A critical role for the peroxisome proliferator-activated receptor alpha (PPARalpha) in the cellular fasting response: the PPARalpha-null mouse as a model of fatty acid oxidation disorders. (7/168)

We hypothesized that the lipid-activated transcription factor, the peroxisome proliferator-activated receptor alpha (PPARalpha), plays a pivotal role in the cellular metabolic response to fasting. Short-term starvation caused hepatic steatosis, myocardial lipid accumulation, and hypoglycemia, with an inadequate ketogenic response in adult mice lacking PPARalpha (PPARalpha-/-), a phenotype that bears remarkable similarity to that of humans with genetic defects in mitochondrial fatty acid oxidation enzymes. In PPARalpha+/+ mice, fasting induced the hepatic and cardiac expression of PPARalpha target genes encoding key mitochondrial (medium-chain acyl-CoA dehydrogenase, carnitine palmitoyltransferase I) and extramitochondrial (acyl-CoA oxidase, cytochrome P450 4A3) enzymes. In striking contrast, the hepatic and cardiac expression of most PPARalpha target genes was not induced by fasting in PPARalpha-/- mice. These results define a critical role for PPARalpha in a transcriptional regulatory response to fasting and identify the PPARalpha-/- mouse as a potentially useful murine model of inborn and acquired abnormalities of human fatty acid utilization.  (+info)

The functions of the flavin contact residues, alphaArg249 and betaTyr16, in human electron transfer flavoprotein. (8/168)

Arg249 in the large (alpha) subunit of human electron transfer flavoprotein (ETF) heterodimer is absolutely conserved throughout the ETF superfamily. The guanidinium group of alphaArg249 is within van der Waals contact distance and lies perpendicular to the xylene subnucleus of the flavin ring, near the region proposed to be involved in electron transfer with medium chain acyl-CoA dehydrogenase. The backbone amide hydrogen of alphaArg249 is within hydrogen bonding distance of the carbonyl oxygen at the flavin C(2). alphaArg249 may modulate the potentials of the two flavin redox couples by hydrogen bonding the carbonyl oxygen at C(2) and by providing delocalized positive charge to neutralize the anionic semiquinone and anionic hydroquinone of the flavin. The potentials of the oxidized/semiquinone and semiquinone/hydroquinone couples decrease in an alphaR249K mutant ETF generated by site directed mutagenesis and expression in Escherichia coli, without major alterations of the flavin environment as judged by spectral criteria. The steady state turnover of medium chain acyl-CoA dehydrogenase and glutaryl-CoA dehydrogenase decrease greater than 90% as a result of the alphaR249Ks mutation. In contrast, the steady state turnover of short chain acyl-CoA dehydrogenase was decreased about 38% when alphaR249K ETF was the electron acceptor. Stopped flow absorbance measurements of the oxidation of reduced medium chain acyl-CoA dehydrogenase/octenoyl-CoA product complex by wild type human ETF at 3 degrees C are biphasic (t(1/2)=12 ms and 122 ms). The rate of oxidation of this reduced binary complex of the dehydrogenase by the alphaR249K mutant ETF is extremely slow and could not be reasonably estimated. alphaAsp253 is proposed to function with alphaArg249 in the electron transfer pathway from medium chain acyl-CoA dehydrogenase to ETF. The steady state kinetic constants of the dehydrogenase were not altered when ETF containing an alphaD253A mutant was the substrate. However, t(1/2) of the rapid phase of oxidation of the reduced medium chain acyl-CoA dehydrogenase/octenoyl-CoA charge transfer complex almost doubled. betaTyr16 lies on a loop near the C(8) methyl group, and is also near the proposed site for interflavin electron transfer with medium chain acyl-CoA dehydrogenase. The tyrosine residue makes van der Waals contact with the C(8) methyl group of the flavin in human ETF and Paracoccus denitrificans ETF (as betaTyr13) and lies at a 30 degrees C angle with the plane of the flavin. Human betaTyr16 was substituted with leucine and alanine residues to investigate the role of this residue in the modulation of the flavin redox potentials and in electron transfer to ETF. In betaY16L ETF, the potentials of the flavin were slightly reduced, and steady state kinetic constants were modestly altered. Substitution of an alanine residue for betaTyr16 yields an ETF with potentials very similar to the wild type but with steady state kinetic properties similar to betaY16L ETF. It is unlikely that the beta methyl group of the alanine residue interacts with the flavin C(8) methyl. Neither substitution of betaTyr16 had a large effect on the fast phase of ETF reduction by medium chain acyl-CoA dehydrogenase.  (+info)

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