Research in this laboratory focuses on a branch of the electron transport system that transfers electrons from at least nine primary flavoprotein dehydrogenases to the main respiratory chain. Four of these dehydrogenases are the chain length specific acyl-CoA dehydrogenases that catalyze the oxidation of acyl-CoA in the first reaction of each cycle of mitochondrial fatty acid b-oxidation. We are investigating the catalytic mechanism of glutary-CoA dehydrogenase (GCD), a tetrameric flavoprotein dehydrogenase that catalyzes the oxidation of glutaryl-CoA, an intermediate in the oxidation of lysine. The electron acceptor for all nine dehydrogenases is electron transfer flavoprotein (ETF) which transfers electrons to a membrane-bound iron-sulfur flavoprotein, ETF-ubiquinone oxidoreductase (ETF-QO). The investigations are driven by the fact that inherited defects in these proteins cause metabolic diseases that are often fatal. Our approach is to identify patients’ mutations in the proteins, and then express and purify the mutant proteins. The proteins are then investigated by a number of biochemical and biophysical methods to determine how the mutations affect the normal function of the proteins.
A key part of these investigations has been the determination of the three dimensional structures of the wild type proteins. At this point, we have solved the crystal structures of GCD, ETF and the structure of ETF-QO is almost complete, with resolution to 2.6. This approach enables to understand on a structural level, how a specific mutation may alter the activity of a protein. For example, the most frequent human mutation in ETF is substitution of a Thr266 by methionine. This mutation disrupts hydrogen bonding of the Thr hydroxyl to the N(5) position of the flavin and lowers the oxidation-reduction potential of the flavin, making it a poor electron acceptor. Mutation of Glu414 in GCD inactivates the dehydrogenase by removing the catalytic base that abstracts the a proton from the substrate, the step that initiates the catalytic pathway. Investigations of pathogenic mutations have provided insight into the normal functions of these enzymes.
Salazar D, Zhang L, deGala G, Frerman FE: Expression and characterization of two pathogenic mutations in human electron transfer flavoprotein. J Biol Chem 272, 26425-26433, 1997.
Roberts DL, Salazar D, Fulmer JP, Frerman FE, Kim J-JP: Crystal structure of Paracoccus denitrificans electron transfer flavoprotein and electrostatic analysis of a conserved flavin binding domain. Biochemistry, 38, 1977-1989, 1999.
Dwyer TM, Mortl S, Kemter K, Bacher A, Fauq A, Frerman FE: The intraflavin hydrogen bond in human electron transfer flavoprotein modulates redox potentials and may participate in electron transfer. Biochemistry, 38, 9735-9745, 1999.
Dwyer,TM, Zhang L, Muller M, Marrugo F, Frerman FE: The roles of the flavin contact residues. a Arg249 and b Tyr16, in human electron transfer flavoprotein. Biochim Biophys Acta, 1433, 139-152, 1999.
Kim J-JP, Wang M, Paschke R, Goodman SI, Biery BJ, Frerman FE. The crystal structure of human glutaryl-CoA dehydrogenase. In: Flavins and Flavoproteins. Ghisla S, Kroneck P, Macheroux P and Sund H; Rudolph Weber Publ., Berlin, in press.
Narkewicz, MR, Moores, RR, Battaglia, FC, Frerman, FE: Ontogeny of serine hydroxymethyltransferase isoenzymes in fetal sheep liver, kidney, and placenta. Molec. Genet. and Med. 68, 473-480,2000.
Dwyer, TM, Rao, KS, Goodman, SI, Frerman, FE: Proton abstraction, steady state kinetics and oxidation-reduction potential of human glutaryl-CoA dehydrogenase. Biochemistry, 39, 11488-11499, 2000.
Dwyer, TM, Rao, KS, Westover, JB, Kim, JJP, Frerman, FE. The role of Arg94 in the oxidation and decarboxylation of glutaryl-CoA dehydrogenase by human glutaryl-CoA dehydrogenase. J Biol Chem, 276,
Chohan, KK, Jones, M, Grossmann, JG, Frerman, FE, Scrutton, NS, Sutcliffe, MJ. Protein dynamics enhance electronic coupling in electron transfer complexes. J Biol Chem, 34142-34147, 2001.
Westover, JB, Goodmamn, SI, Frerman, FE. The binding, hydration and decarboxylation of the reaaction intermediate, glutaconyl-CoA by human glutaryl-CoA dehydrogenase. Biochemistry, in press, 2001.
Rao, SK, Albro, M, Vockley, J, Frerman, FE. Mechanism-based inactivation of human glutaryl-CoA dehydrogenase by 2-pentynoyl-CoA: Rationale for enhanced reactivity. J Biol Chem, 278, 26342-26350, 2003.
Simkovic, M, Frerman, FE. Alternate quinone substrates and inhibitors of human electron flavoprotein-ubiquinone oxidoreductase. Biochem J., 378, 633-640, 2003.
Fu, Z., Wang, M., Paschke, R., Rao, K.S., Frerman, F.E., and Kim, J.J. Crystal structure of human glutaryl-CoA dehydrogenase with and without an alternate substrate: structural bases of dehydrogenation and decarboxylation. Biochemistry 43, 9674-9684, 2004.