MOLECULAR CHARACTERIZATION OF ACYL-COA DEHYDROGENASES

Summary

Principal Investigator: Jerry Vockley
Affiliation: Children's Hospital of Pittsburgh
Country: USA
Abstract: The acyl-CoA dehydrogenases (ACDs) are a family of evolutionarily related enzymes involved in the first step of the beta-oxidation of fatty acids and in the intermediate metabolism of leucine, isoleucine and valine. Deficiencies of these enzymes are increasingly being recognized as important causes of inherited defects of metabolism in humans. The long range objective of this application is to investigate important structure/ function relationships in the ACD gene family at the molecular level, and relate this information to mutations responsible for clinical deficiencies of these enzymes. Specific aims of this proposal include 1) analysis of the pathway of maturation of isovaleryl-CoA dehydrogenase (IVD), a member of this gene family, focusing on the process of tetramerization, 2) expression analysis of mutant IVD alleles from patients with deficiencies of IVD as a source of naturally occurring mutations of functional significance, and 3) characterization of structure/function relationships important in tetramerization of IVD and determination of substrate specificity of other ACD family members. The pathway of IVD maturation will be studied by metabolic labeling of IVD overexpressed in tissue culture cells via a eukaryotic expression vector, and by in vitro mitochondrial processing experiments. Cell fractionation studies will be used to identify the subcellular localization of this process, and gel filtration of mitochondrial extracts from in vitro transport experiments will identify intra-mitochondrial intermediates. In vitro mutagenesis will be used to produce IVD mutants deficient in tetramerization in order to identify amino acid motifs and residues critical to this process. Mutant alleles from patients with deficiencies of IVD will be studied via these same techniques in order to characterize the functional nature of the abnormal enzyme proteins produced by these patients' cells. Finally, structure/function relationships within this gene family will be dissected in a more rigorous fashion using the techniques of PCR in vitro site- specific mutagenesis and domain switching. These investigations will focus on the nature of the amino acid residues controlling the determination of substrate specificity of the various ACDs. Normal short and long chain acyl-CoA dehydrogenases will be expressed in a prokaryotic expression systems, and SCAD tertiary and quarternery structure will be studied by x- ray crystallography. Domain switching techniques will be used to synthesize hybrid ACDs with altered substrate binding specificities, and the substrate specificity will be correlated to the ACD sequences present to identify amino acid sequences important in determining this characteristic. These studies will lead to a more complete understanding of the ACD gene family, and an improvement in the ability to diagnose and treat patients with these disorders.
Funding Period: 1993-08-01 - 1996-07-31
more information: NIH RePORT

Top Publications

  1. ncbi Convergent evolution of a 2-methylbutyryl-CoA dehydrogenase from isovaleryl-CoA dehydrogenase in Solanum tuberosum
    Eric S Goetzman
    Department of Pediatrics, School of Medicine and Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh, Children s Hospital of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA
    J Biol Chem 280:4873-9. 2005
  2. ncbi Different spectrum of mutations of isovaleryl-CoA dehydrogenase (IVD) gene in Korean patients with isovaleric acidemia
    Yong Wha Lee
    Department of Laboratory Medicine and Genetics, Soonchunhyang University Bucheon Hospital, Soonchunhyang University College of Medicine, Bucheon, Republic of Korea
    Mol Genet Metab 92:71-7. 2007
  3. pmc A new genetic disorder in mitochondrial fatty acid beta-oxidation: ACAD9 deficiency
    M He
    Children s Hospital of Pittsburgh, Department of Pediatrics, University of Pittsburgh, School of Medicine, Pittsburgh, PA, 15213, USA
    Am J Hum Genet 81:87-103. 2007
  4. pmc Expression and characterization of mutations in human very long-chain acyl-CoA dehydrogenase using a prokaryotic system
    Eric S Goetzman
    Department of Pediatrics, School of Medicine, University of Pittsburgh, Children s Hospital of Pittsburgh, PA 15213, USA
    Mol Genet Metab 91:138-47. 2007
  5. ncbi In vitro characterization and in vivo expression of human very-long chain acyl-CoA dehydrogenase
    J Lawrence Merritt
    Department of Medical Genetics, Mayo Clinic College of Medicine, Rochester, MN, USA
    Mol Genet Metab 88:351-8. 2006
  6. pmc Isovaleric acidemia: new aspects of genetic and phenotypic heterogeneity
    Jerry Vockley
    Department of Pediatrics, University of Pittsburgh School of Medicine, Children s Hospital of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, PA 15238, USA
    Am J Med Genet C Semin Med Genet 142:95-103. 2006
  7. ncbi Systemic correction of a fatty acid oxidation defect by intramuscular injection of a recombinant adeno-associated virus vector
    Thomas J Conlon
    Department of Pediatrics, Powell Gene Therapy Center, University of Florida, Gainesville, FL 32610, and Department of Pediatrics, Children s Hospital of Pittsburgh, PA, USA
    Hum Gene Ther 17:71-80. 2006
  8. ncbi Functional analysis of acyl-CoA dehydrogenase catalytic residue mutants using surface plasmon resonance and circular dichroism
    Eric S Goetzman
    Department of Pediatrics, School of Medicine, University of Pittsburgh, Children s Hospital of Pittsburgh, Pittsburgh, PA, USA
    Mol Genet Metab 87:233-42. 2006
  9. pmc Medium-chain acyl-CoA dehydrogenase deficiency in gene-targeted mice
    Ravi J Tolwani
    Department of Genetics, University of Alabama, Birmingham, Alabama, USA
    PLoS Genet 1:e23. 2005
  10. ncbi In vitro correction of medium chain acyl CoA dehydrogenase deficiency with a recombinant adenoviral vector
    David B Schowalter
    Department of Medical Genetics, Mayo Clinic College of Medicine, Rochester, MN, USA
    Mol Genet Metab 85:88-95. 2005

Scientific Experts

  • I Tein
  • David B Schowalter
  • Jerry Vockley
  • Eric S Goetzman
  • Dietrich Matern
  • Yong Wha Lee
  • M He
  • Miao He
  • Tien V Nguyen
  • J Lawrence Merritt
  • Thomas J Conlon
  • Ravi J Tolwani
  • G Murdoch
  • C A Palmer
  • You Kyoung Lee
  • Chang Seok Ki
  • Nam Doo Kim
  • R D Nicholls
  • Dong Hwan Lee
  • Al Walid Mohsen
  • S L Rutledge
  • Z Pei
  • P A Watkins
  • Yudong Wang
  • Brittani K Ninness
  • N Majumder
  • D R Kelly
  • Renius Owen
  • Eric Goetzman
  • Glenn Walter
  • Kirsten Erger
  • Jan Daniels
  • Greg Gutierrez
  • Travis Cossette
  • Terence R Flotte
  • Liqun Tian
  • Piero Rinaldo
  • Doug A Hamm
  • J Daniel Sharer
  • Al Walid A Mohsen
  • Trenton R Schoeb
  • Philip A Wood
  • Kavita Prasad

Detail Information

Publications11

  1. ncbi Convergent evolution of a 2-methylbutyryl-CoA dehydrogenase from isovaleryl-CoA dehydrogenase in Solanum tuberosum
    Eric S Goetzman
    Department of Pediatrics, School of Medicine and Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh, Children s Hospital of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA
    J Biol Chem 280:4873-9. 2005
    ....
  2. ncbi Different spectrum of mutations of isovaleryl-CoA dehydrogenase (IVD) gene in Korean patients with isovaleric acidemia
    Yong Wha Lee
    Department of Laboratory Medicine and Genetics, Soonchunhyang University Bucheon Hospital, Soonchunhyang University College of Medicine, Bucheon, Republic of Korea
    Mol Genet Metab 92:71-7. 2007
    ..0% of control) in all samples. These results confirm IVD mutations in Korean patients with IVA and reveal that the mutation spectrum is different from previously reported patients...
  3. pmc A new genetic disorder in mitochondrial fatty acid beta-oxidation: ACAD9 deficiency
    M He
    Children s Hospital of Pittsburgh, Department of Pediatrics, University of Pittsburgh, School of Medicine, Pittsburgh, PA, 15213, USA
    Am J Hum Genet 81:87-103. 2007
    ....
  4. pmc Expression and characterization of mutations in human very long-chain acyl-CoA dehydrogenase using a prokaryotic system
    Eric S Goetzman
    Department of Pediatrics, School of Medicine, University of Pittsburgh, Children s Hospital of Pittsburgh, PA 15213, USA
    Mol Genet Metab 91:138-47. 2007
    ..In summary, the bacterial expression system developed here will significantly advance our understanding of both the clinical aspects of VLCAD deficiency and the basic biochemistry of the enzyme...
  5. ncbi In vitro characterization and in vivo expression of human very-long chain acyl-CoA dehydrogenase
    J Lawrence Merritt
    Department of Medical Genetics, Mayo Clinic College of Medicine, Rochester, MN, USA
    Mol Genet Metab 88:351-8. 2006
    ..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...
  6. pmc Isovaleric acidemia: new aspects of genetic and phenotypic heterogeneity
    Jerry Vockley
    Department of Pediatrics, University of Pittsburgh School of Medicine, Children s Hospital of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, PA 15238, USA
    Am J Med Genet C Semin Med Genet 142:95-103. 2006
    ..A better understanding of the heterogeneity of this disease and the relevance of genotype/phenotype correlations to clinical management of patients are among the challenges remaining in the study of this disorder in the coming years...
  7. ncbi Systemic correction of a fatty acid oxidation defect by intramuscular injection of a recombinant adeno-associated virus vector
    Thomas J Conlon
    Department of Pediatrics, Powell Gene Therapy Center, University of Florida, Gainesville, FL 32610, and Department of Pediatrics, Children s Hospital of Pittsburgh, PA, USA
    Hum Gene Ther 17:71-80. 2006
    ..This study is the first to demonstrate the systemic correction of a fatty acid oxidation disorder with rAAV and the utility of MRS as a noninvasive method to monitor SCAD correction after in vivo gene therapy...
  8. ncbi Functional analysis of acyl-CoA dehydrogenase catalytic residue mutants using surface plasmon resonance and circular dichroism
    Eric S Goetzman
    Department of Pediatrics, School of Medicine, University of Pittsburgh, Children s Hospital of Pittsburgh, Pittsburgh, PA, USA
    Mol Genet Metab 87:233-42. 2006
    ..Lastly, competitive inhibition studies using the ETF fluorescence reduction assay suggested that SCAD E368G and IVD E254G do not effectively compete with the wild-type enzymes for the physiological electron acceptor ETF...
  9. pmc Medium-chain acyl-CoA dehydrogenase deficiency in gene-targeted mice
    Ravi J Tolwani
    Department of Genetics, University of Alabama, Birmingham, Alabama, USA
    PLoS Genet 1:e23. 2005
    ..The MCAD-deficient mouse reproduced important aspects of human MCAD deficiency and is a valuable model for further analysis of the roles of fatty acid oxidation and pathogenesis of human diseases involving fatty acid oxidation...
  10. ncbi In vitro correction of medium chain acyl CoA dehydrogenase deficiency with a recombinant adenoviral vector
    David B Schowalter
    Department of Medical Genetics, Mayo Clinic College of Medicine, Rochester, MN, USA
    Mol Genet Metab 85:88-95. 2005
    ....
  11. ncbi Short-chain acyl-CoA dehydrogenase gene mutation (c.319C>T) presents with clinical heterogeneity and is candidate founder mutation in individuals of Ashkenazi Jewish origin
    Ingrid Tein
    Division of Neurology, Department of Pediatrics, Laboratory Medicine and Pathobiology, Hospital for Sick Children, University of Toronto, Toronto, Canada M5G 1X8
    Mol Genet Metab 93:179-89. 2008
    ..This should be screened for in individuals with multicore myopathy, particularly among the Ashkenazim...