The mammalian branched-chain dehydrogenase complex (abbreviated branched- chain complex) is a mitochondrial multienzyme system that catalyzes the oxidative decarboxylation of alpha-keto acids derived from the branched-chain amino acids leucine, isoleucine and valine. The enzyme complex organized about a transacylase (E2b) core, consisting of 24 lipoyl-bearing polypeptides, to which a decarboxylase (Elb), a dehydrogenase (E3), a specific kinase and a specific phosphatase are attached through non-covalent interactions. The long-range goal of this project is to understand at the molecular level the structure and function of the mammalian branched-chain complex. The information obtained will have wide implications on macro-molecular organization and function.
Specific aims are: 1) To determine the crystal structure of the E2b inner core. X-ray crystallography will be carried out with highly purified recombinant E2b proteins. The crystal structure of the inner-core domain when solved will be compared with that of E. coli chloramphenicol acetyltransferase type III. This approach will test the hypothesis that the three-dimensional structures of acyl-CoA transferases are conserved during evolution. 2) To define the active site and subunit-binding domains of E2b. Site-directed mutagenesis and serial deletion experiments will be utilized to map the active site and subunit-binding region of E2b. These experiments are aimed to establish His 391 as the active site for the transacylation reaction. Methods will be developed to establish the putative Elb/E3 binding domain. Flexible hinge regions in recombinant E2b will also be studied by 1H-NMR spectroscopy. 3) To investigate the mammalian lipoylation system. We will use a lipoylation assay to locate the subcellular compartment in which the apo-E2b is lipoylated, and to purify and characterize the mammalian lipoylating enzyme. These experiments will test the hypothesis that lipoylation occurs inside mitochondria. 4) To study structure and function of the Elb-component. Recombinant Elb-alpha and Elb-beta subunits will be utilized to study their assembly and their ability to bind and reductively acylate lipoyl-E2b. The putative TPP-binding site on Elb-alpha will be established by site-directed mutagenesis and equilibrium dialysis. These experiments will define the catalytic and structural roles of Elb-alpha and beta-subunits in the branched-chain complex.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK037373-04A1
Application #
3236240
Study Section
Biochemistry Study Section (BIO)
Project Start
1989-01-01
Project End
1995-05-31
Budget Start
1991-06-01
Budget End
1992-05-31
Support Year
4
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Type
Schools of Medicine
DUNS #
City
Dallas
State
TX
Country
United States
Zip Code
75390
Kennedy, L; Bittel, D C; Kibiryeva, N et al. (2006) Circulating adiponectin levels, body composition and obesity-related variables in Prader-Willi syndrome: comparison with obese subjects. Int J Obes (Lond) 30:382-7
Davie, J R; Wynn, R M; Meng, M et al. (1995) Expression and characterization of branched-chain alpha-ketoacid dehydrogenase kinase from the rat. Is it a histidine-protein kinase? J Biol Chem 270:19861-7
Chuang, D T; Davie, J R; Wynn, R M et al. (1995) Molecular basis of maple syrup urine disease and stable correction by retroviral gene transfer. J Nutr 125:1766S-1772S
Wynn, R M; Davie, J R; Zhi, W et al. (1994) In vitro reconstitution of the 24-meric E2 inner core of bovine mitochondrial branched-chain alpha-keto acid dehydrogenase complex: requirement for chaperonins GroEL and GroES. Biochemistry 33:8962-8
Wynn, R M; Davie, J R; Cox, R P et al. (1994) Molecular chaperones: heat-shock proteins, foldases, and matchmakers. J Lab Clin Med 124:31-6
Meng, M; Chuang, D T (1994) Site-directed mutagenesis and functional analysis of the active-site residues of the E2 component of bovine branched-chain alpha-keto acid dehydrogenase complex. Biochemistry 33:12879-85
Wynn, R M; Kochi, H; Cox, R P et al. (1994) Differential processing of human and rat E1 alpha precursors of the branched-chain alpha-keto acid dehydrogenase complex caused by an N-terminal proline in the rat sequence. Biochim Biophys Acta 1201:125-8
Lau, K S; Herring, W J; Chuang, J L et al. (1992) Structure of the gene encoding dihydrolipoyl transacylase (E2) component of human branched chain alpha-keto acid dehydrogenase complex and characterization of an E2 pseudogene. J Biol Chem 267:24090-6
Lau, K S; Chuang, J L; Herring, W J et al. (1992) The complete cDNA sequence for dihydrolipoyl transacylase (E2) of human branched-chain alpha-keto acid dehydrogenase complex. Biochim Biophys Acta 1132:319-21
Wynn, R M; Davie, J R; Cox, R P et al. (1992) Chaperonins groEL and groES promote assembly of heterotetramers (alpha 2 beta 2) of mammalian mitochondrial branched-chain alpha-keto acid decarboxylase in Escherichia coli. J Biol Chem 267:12400-3

Showing the most recent 10 out of 29 publications