Research Interests

Catalyic Mechanism and Biological Roles of Succinyl-CoA Synthetase and Related Enzymes (funded by NSERC)

Biochemistry students are introduced to succinyl-CoA synthetase (SCS) as one of the enzymes of the citric acid cycle. In the citric acid cycle, SCS catalyzes the reaction that uses a molecule of succinyl-CoA and inorganic phosphate to phosphorylate ADP or GDP; and releases succinate and CoA. The enzyme is interesting because

• it participates in the reaction through the phosphorylation of a histidine residue,
• it has two different subunits, named alpha and beta, and some forms of SCS are alpha,beta-dimers while others are alpha₂beta₂-tetramers,
• some forms of SCS can use either ADP or GDP while other forms can use only one of the two,
• the enzyme must bind three substrates and releases three products

The goal of our work is to understand how SCS catalyzes the reaction. As we learn about SCS, we gain an understanding of other multi-subunit proteins, of other enzymes that catalyze reactions of nucleotides, and of other proteins that are phosphorylated on histidine residues.

We solved the structure of E. coli SCS using X-ray crystallography. In this structure, the active site histidine residue is phosphorylated. The structure showed that the alpha-subunit binds CoA with the reactive group of CoA located near the phosphorylated histidine residue. The phosphorylated histidine residue is stabilized by interactions with both the alpha- and the beta-subunits. We later discovered that the nucleotide, ADP, binds in another domain of the beta-subunit, ~35 Å away from the active site histidine residue. We think that the loop that includes this histidine residue can flip to position the histidine residue near the nucleotide-binding site. We are now trying to prove this hypothesis.  Other projects are directed at determining the binding site for succinate and at understanding how different forms of SCS discriminate between nucleotides. It is suspected that the different forms of SCS are needed for the enzyme to perform its various functions. In addition to its role in the citric acid cycle, SCS produces succinyl-CoA for ketone body metabolism and for porphyrin synthesis.

Related enzymes include ATP-citrate lyase, which catalyzes the conversion of citrate and CoA into acetyl-CoA and oxaloacetate, coupled with the hydrolysis of ATP.  ATP-citrate lyase is the cytoplasmic enzyme linking energy metabolism from carbohydrates to the production of fatty acids.  Since this link is important in many cancer cells, the enzyme is a possible target for drug design.

 

Courses Taught

Graduate Students

Awards

2007 - AHFMR Medical Scholar- salary award
2002 - NSERC (1999-02) Univ Faculty Award, one of the top three candidates in the Ist year of the program 

Selected publications

• Fraser, M.E., Hayakawa, K., Brown, W.D. (2010) Catalytic Role of the Conformational Change in Succinyl-CoA:3-Oxoacid CoA Transferase on Binding CoA. Biochemistry Oct 26. [Epub ahead of print]  PubMed Abstract. PDB Identifier: 3OXO.
  
• Sun, T., Hayakawa, K., Bateman, K.S., Fraser, M.E. (2010) Identification of the Citrate-Binding Site of Human ATP-Citrate Lyase using X-ray Crystallography.  Journal of Biological Chemistry 285, 27418-28.  PubMed Abstract.  PDB Identifiers: 3MWD, 3MWE.
• Yamniuk, Y.P., Anderson, K.L., Fraser, M.E., Vogel, H.J. (2009) Auxilliary Ca2+ binding sites can influence the structure of CIB1.  Protein Science 18, 1128-34.   PubMed Abstract. 
• Tammam, S.D., Rochet, J.-C., Fraser, M.E. (2007) Identification of the Cysteine Residue Exposed by the Conformational Change in Pig Heart Succinyl-CoA:3-Ketoacid Coenzyme A Transferase on Binding CoA.  Biochemistry 46 , 10852 - 10863 .  PubMed Abstract.   PDF.  PDB Identifiers: 2NRB, 2NRC.
  
• Hidber, E., Brownie, E.R., Hayakawa, K., Fraser, M.E. (2007) Participation of Cys 123α of E. coli Succinyl-CoA Synthetase in Catalysis. Acta Crystallographica D63, 876-884. PubMed Abstract.  PDF.  PDB Identifiers: 2NU6, 2NU7, 2NU8, 2NU9, 2NUA.
• Joyce, M.A., Brownie, E.R., Hayakawa, K., Fraser, M.E. (2007) Cloning, Expression, Purification, Crystallization and Preliminary X-ray Analysis of Thermus aquaticus Succinyl-CoA Synthetase. Acta Crystallographica F63, 399-402.  PubMed Abstract.  PDF.
  
• Sutendra, G., Wong, S., Fraser, M.E., Huber, R.E. (2007) b-Galactosidase (Escherichia coli) Has a Second Catalytically Important Mg2+ Site. Biochemical and Biophysical Research Communications 352, 566-570. PubMed Abstract.
• Fraser, M.E., Cherney, M.M., Marcato, P., Mulvey, G.L., Armstrong, G.D., James, M.N.G. (2006) Binding of Adenine to Stx2, the Protein Toxin from E. coli O157:H7. Acta Crystallographica F62, 627-630. PubMed Abstract.  PDF.  PDB Identifiers: 2GA4.
• Luo, L., Pappalardi, M.B., Tummino, P.J., Copeland, R.A., Fraser, M.E., Grzyska, P.K., Hausinger, R.P. (2006) An Assay for Fe(II)/2-oxoglutarate-dependent Dioxygenases by Enzyme-coupled Detection of Succinate Formation. Analytical Biochemistry 353, 69-74. PubMed Abstract.
• Fraser, M.E., Hayakawa, K., Hume, M.S., Ryan, D.G., Brownie, E.R. (2006) Interactions of GTP with the ATP-grasp Domain of GTP-specific Succinyl-CoA Synthetase.  Journal of Biological Chemistry 281, 11058-11065. PubMed Abstract. PDB Identifiers: 2FPI, 2FPP, 2FP4, 2FPG.
• Rangarajan, E.S., Li, Y., Ajamian, E., Iannuzzi, P., Kernaghan, S.D., Fraser, M.E., Cygler, M., Matte, A. (2005) Crystallographic Trapping of the Glutamyl-CoA Thioester Intermediate of Family I CoA Transferases.  Journal of Biological Chemistry 280, 42919-42928. PubMed Abstract. PDB Identifiers: 2AHU, 2AHV, 2AHW.
• Bridges, D., Fraser, M.E., Moorhead, G.B. (2005) Cyclic Nucleotide Binding Proteins in the Arabidopsis thaliana and Oryza sativa Genomes, BMC Bioinformatics 6, 6. PubMed Abstract.
• Coros, A.M., Swenson, L., Wolodko, W.T, Fraser, M.E. (2004) Structure of the CoA Transferase from Pig Heart to 1.7 Å Resolution, Acta Crystallographica Section D 60, 1717-1725. PubMed Abstract. PDB Identifiers: 1OOY, 1OOZ, 1OPE.
• Fraser, M.E., Fujinaga, M., Cherney, M.M., Melton-Celsa, A.R., Twiddy, E.M., O'Brien, A.D., James, M.N.G. (2004) Structure of Shiga Toxin Type 2 (Stx2) from Escherichia coli O157:H7, Journal of Biological Chemistry 279, 27511-27517. PubMed Abstract. PDB Identifiers: 1R4P, 1R4Q.