This proposal is to extend the structural studies on the medium chain acyl- CoA dehydrogenase (MCAD) of pig liver to obtain a more detailed picture of this enzyme and to determine structures of related enzymes, the human MCAD, the porcine short chain acyl CoA dehydrogenase (SCAD) and a similar enzyme from ma bacterium. These are members of a family of enzymes that carry out the first oxidative step in the catabolism of fatty acids, the principal fuel for many organs, including liver, kidney, heart and skeletal muscle. The rate o oxidation can be altered by diet, physiological state and disease, exemplified by starvation, pregnancy and diabetes. The critical role of these enzymes in metabolism is illustrated by the severity of human diseases attributed to inherited deficiencies of each of the three dehydrogenases. The MCAD from pig liver mitochondria has recently been studies intensively and progress has been made in elucidating the catalytic, structural and cellular properties of the enzyme. Detailed structural information from high resolution X-ray analysis will enable us to relate chemical functions to the structure of the protein and to define the catalytic mechanism. The enzyme has a molecular weight of 172,000 and contains four identical subunits, each containing one equivalent of flavin adenine dinucleotide (FAD). Three-dimensional X-ray analysis of the enzyme at 3A resolution has revealed the location and orientation of the FAD and the conformation of the entire peptide chain. The polypeptide folding near the FAD binding site is different from the structures seen in other flavoproteins. It is proposed to extend the structural analysis to 2A resolution and to elucidate the detailed mechanism of oxidation of acyl-CoA thioesters and transfer of electrons to the electron transfer flavoprotein, the physiological oxidant of he dehydrogenase in mitochondria. It is also proposed to initiate both enzymatic and crystallographic studies on the human MCAD; this enzyme has been cloned and the studies will employ both the native protein and altered proteins made by site-directed mutagenesis. Crystallographic studies will also be carried out on the SCAD of pig liver and on the butyryl-CoA dehydrogenase of M. elsdenii, which has already been crystallized in a form suitable for X-ray analysis. Comparisons of these structures will enable us to ascertain the structures involved in the general catalytic mechanisms of this group of enzymes and the features that determine the specificity of each acyl-CoA dehydrogenase.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029076-11
Application #
3276553
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1982-03-01
Project End
1995-06-30
Budget Start
1993-07-01
Budget End
1994-06-30
Support Year
11
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Medical College of Wisconsin
Department
Type
Schools of Medicine
DUNS #
073134603
City
Milwaukee
State
WI
Country
United States
Zip Code
53226
Floyd, Brendan J; Wilkerson, Emily M; Veling, Mike T et al. (2016) Mitochondrial Protein Interaction Mapping Identifies Regulators of Respiratory Chain Function. Mol Cell 63:621-632
Schiff, Manuel; Haberberger, Birgit; Xia, Chuanwu et al. (2015) Complex I assembly function and fatty acid oxidation enzyme activity of ACAD9 both contribute to disease severity in ACAD9 deficiency. Hum Mol Genet 24:3238-47
Fu, Zhuji; Runquist, Jennifer A; Montgomery, Christa et al. (2010) Functional insights into human HMG-CoA lyase from structures of Acyl-CoA-containing ternary complexes. J Biol Chem 285:26341-9
Gobin-Limballe, St├ęphanie; McAndrew, Ryan P; Djouadi, Fatima et al. (2010) Compared effects of missense mutations in Very-Long-Chain Acyl-CoA Dehydrogenase deficiency: Combined analysis by structural, functional and pharmacological approaches. Biochim Biophys Acta 1802:478-84
Fu, Zhuji; Voynova, Natalia E; Herdendorf, Timothy J et al. (2008) Biochemical and structural basis for feedback inhibition of mevalonate kinase and isoprenoid metabolism. Biochemistry 47:3715-24
McAndrew, Ryan P; Wang, Yudong; Mohsen, Al-Walid et al. (2008) Structural basis for substrate fatty acyl chain specificity: crystal structure of human very-long-chain acyl-CoA dehydrogenase. J Biol Chem 283:9435-43
Tu, Xi; Hubbard, Paul A; Kim, Jung-Ja P et al. (2008) Two distinct proton donors at the active site of Escherichia coli 2,4-dienoyl-CoA reductase are responsible for the formation of different products. Biochemistry 47:1167-75
McAndrew, R P; Vockley, J; Kim, J-J P (2008) Molecular basis of dimethylglycine dehydrogenase deficiency associated with pathogenic variant H109R. J Inherit Metab Dis 31:761-8
Gobin-Limballe, S; Djouadi, F; Aubey, F et al. (2007) Genetic basis for correction of very-long-chain acyl-coenzyme A dehydrogenase deficiency by bezafibrate in patient fibroblasts: toward a genotype-based therapy. Am J Hum Genet 81:1133-43
Rao, K Sudhindra; Fu, Zhuji; Albro, Mark et al. (2007) The effect of a Glu370Asp mutation in glutaryl-CoA dehydrogenase on proton transfer to the dienolate intermediate. Biochemistry 46:14468-77

Showing the most recent 10 out of 44 publications