Defects in the flavoprotein, glutaryl-CoA dehydrogenase (GCD), cause glutaric aciduria type I (GA1), an autosomal recessively inherited neurometabolic disorder. GCD catalyzes the alpha, beta dehydrogenation of glutaryl-CoA by a mechanism which is common along acyl-CoA dehydrogenases. GCD also catalyzes the decarboxylation of the enzyme- bound intermediate, glutaconyl-CoA, to crotonyl-CoA and CO2. Decarboxylation of glutaconyl-CoA requires oxidation of the dehydrogenase flavin and protonation of the proposed crotonyl-CoA anion (-CH2=-CH=-CH=-COSCoA). In patients with defects in GCD, the onset of neurological symptoms in GA1 patients usually follows a viral injection early in life. Over 50 missense mutations have been identified that may affect oxidation and decarboxylation of glutaryl-CoA. These mutations may also affect assembly or stability of the tetramer, the oxidation-reduction potential of the dehydrogenase flavin or reoxidation of the dehydrogenase flavin by electron transfer flavoprotein (ETF). The proposed research has the following specific aims. [1] A number of mutant alleles will be expressed and the defective proteins characterized by kinetic and redox methods to access the basis of the enzymatic defects. [2] We will investigate the coupling of glutaryl-CoA oxidation with decarboxylation/protonation of the enzyme bound intermediate, glutaconyl-CoA, using site directed mutations hypothesized to uncouple these steps in catalysis. The crystal structure of GCD with a bound glutaryl-CoA analog and with the reaction intermediate, glutaconyl-CoA, will be determined to provide insight into the decarboxylation reaction. [3] We will investigate the decarboxylation of glutaconyl-CoA directly and define the roles of specific amino acids in the reaction. The participation of the 2'-hydroxyl of the ribityl side chain of the FAD prosthetic group in stabilization of decarboxylation/protonation intermediates will also be determined.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS039339-04
Application #
6625462
Study Section
Special Emphasis Panel (ZRG1-BIO (02))
Program Officer
Tagle, Danilo A
Project Start
1999-12-10
Project End
2004-11-30
Budget Start
2002-12-01
Budget End
2004-11-30
Support Year
4
Fiscal Year
2003
Total Cost
$277,683
Indirect Cost
Name
University of Colorado Denver
Department
Pediatrics
Type
Schools of Medicine
DUNS #
041096314
City
Aurora
State
CO
Country
United States
Zip Code
80045
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
Rao, K Sudhindra; Albro, Mark; Dwyer, Timothy M et al. (2006) Kinetic mechanism of glutaryl-CoA dehydrogenase. Biochemistry 45:15853-61
Rao, K Sudhindra; Albro, Mark; Zirrolli, Joseph A et al. (2005) Protonation of crotonyl-CoA dienolate by human glutaryl-CoA dehydrogenase occurs by solvent-derived protons. Biochemistry 44:13932-40
Fu, Zhuji; Wang, Ming; Paschke, Rosemary et al. (2004) Crystal structures of human glutaryl-CoA dehydrogenase with and without an alternate substrate: structural bases of dehydrogenation and decarboxylation reactions. Biochemistry 43:9674-84
Rao, K Sudhindra; Albro, Mark; Vockley, Jerry et al. (2003) Mechanism-based inactivation of human glutaryl-CoA dehydrogenase by 2-pentynoyl-CoA: rationale for enhanced reactivity. J Biol Chem 278:26342-50
Westover, Jonna B; Goodman, Stephen I; Frerman, Frank E (2003) Pathogenic mutations in the carboxyl-terminal domain of glutaryl-CoA dehydrogenase: effects on catalytic activity and the stability of the tetramer. Mol Genet Metab 79:245-56
Rao, K Sudhindra; Vander Velde, David; Dwyer, Timothy M et al. (2002) Alternate substrates of human glutaryl-CoA dehydrogenase: structure and reactivity of substrates, and identification of a novel 2-enoyl-CoA product. Biochemistry 41:1274-84
Westover, J B; Goodman, S I; Frerman, F E (2001) Binding, hydration, and decarboxylation of the reaction intermediate glutaconyl-coenzyme A by human glutaryl-CoA dehydrogenase. Biochemistry 40:14106-14
Dwyer, T M; Rao, K S; Westover, J B et al. (2001) The function of Arg-94 in the oxidation and decarboxylation of glutaryl-CoA by human glutaryl-CoA dehydrogenase. J Biol Chem 276:133-8
Dwyer, T M; Rao, K S; Goodman, S I et al. (2000) Proton abstraction reaction, steady-state kinetics, and oxidation-reduction potential of human glutaryl-CoA dehydrogenase. Biochemistry 39:11488-99