We are interested in understanding the mechanism and regulation of enzymes. This includes, dissecting the contributions of substrate binding and protein-protein interactions to catalysis, determining the structure of the transition state and the mechanism of substrate selectivity. Our research integrates a variety of techniques from molecular biology, protein expression and purification, steady and pre-steady state kinetics, computational modeling, x-ray crystallography, and UV/visible and fluorescence spectroscopies. The following projects are currently being developed in the laboratory.

1) Mechanism of hydroxylation of siderophores in microbial pathogens. During infection many human pathogens produce and secrete low molecular weight peptidic metabolites called, siderophores. The role of siderophores is to scavenge ferric iron from the host in order for the bacteria to proliferate. We are interested in the biosynthesis of the siderophores produced by the human pathogens Mycobacterium tuberculosis, Aspergillus fumigatus, and Yersinia pestis. Specifically, we are targeting the enzymes that hydroxylate the siderophores to form a hydroxamate moiety, which is essential for iron binding. Our objective is to provide a detailed understanding of the chemical and kinetic mechanisms and to determine the 3-dimensional structure for this novel family of enzymes.

2) UDP-Galactopyranose mutase reaction. Galactofuranose (Galf) is an important component of the cell surface of several pathogenic bacteria, protozoan, fungi and mycobacterium. Galf is not present in humans, making its biosynthetic pathway a target for new antibiotics. Galf is produced from the transformation of UDP-galactopyranose to UDP-galactofuranose by the flavin-containing enzyme, UDP-glactopyranose mutase (UGM). We are interested in understanding the role of the flavin cofactor in catalysis, determining the 3-dimensional structure and to identify novel inhibitors against the UGM from the human pathogens: M. tuberculosis, L. major, T. cruzi and A. fumigatus.

3) Bacterial multicomponent diiron monooxygenases. Bacterial multicomponent monooxygenases (BMM) are a family of nonheme diiron enzymes involved in the hydroxylation of a variety of hydrocarbons. The catalytic cycle of these enzymes consists of substrate binding, electron transfer from NAD H to the diiron site to form a diferrous center, and O2 activation prior to substrate hydroxylation. There are three conserved components involved in this cycle: 1) a hydroxylase complex, composed of at least a large catalytic alpha and small structural beta subunits and, in some cases, an additional gamma subunit, 2) an oxidoreductase containing a ferredoxin like domain and a NAD H-dependent reductase domain, 3) a cofactorless regulatory protein involved in coupling the oxidation of NAD H to the hydroxylation of the organic substrate. We are studying the uncharacterized members of Group 5 diiron monooxygenases. Specifically, we are trying to elucidate the role of protein-protein interactions in oxygen activation and substrate selectivity.

4) a) Determination of the activity, regulation and identification of interacting partners of mammalian casein kinase 1 splice variants and b) Characterization of CK1 as a target for anti-parasitic chemotherapy. a) Casein kinase I (CK1) is a serine-threonine kinase that catalyzes the phosphorylation of many proteins that play important roles in cell division, differentiation, circadian rhythms, and metabolic control. Mammalian CK1 has four splice variants differing in the presence or absence of 28 amino acids (L insert) in the catalytic domain and 12 amino acids at the C-terminus. The presence or absence of these inserts produce four isoforms: CK1, CK1-L, CK1-LS, and CK1-S. The physiological function of the CK1 splice variants is not well understood. We are using cross-linking strategies to identify proteins that associate and modulate the function of these kinases. Additionally, we are using protein engineering and substrate analogues in combination with mass spectrometry to identify novel substrates.
b) CK1 is found in the human protozoan parasites, plasmodium, trypanosoma and leishmania. These enzymes have been shown to be important in pathogenesis. Our research interest is in the characterization of all three CK1 isoforms and the identification of their substrates. Our studies are aimed to determine the role of these enzymes in pathogenesis.

Sobrado, P., Gorem, M.A., James, D., Amundson, C.K., Fox, B.G. (2008) A Protein Structure Initiative approach to expression, purification, and in situ delivery of human cytochrome b5 to membrane vesicles. Protein Expr. Purif. 2. 229-41    [Abstract]

Sobrado, P. (2008) Functional Expression and Purification of UDP-Galactopyranose Mutase from Trypanosoma cruzi. In Flavins and Flavoproteins (Frago, S., Gomez-Moreno, C, Medina, M. eds.) 509-513.

Tsai, C-L., Gokulan, K., Sobrado, P., Sacchettini, J.C. and Fitzpatrick, F.P.(2007) Mechanistic and Structural Studies of H373Q Flavocytochrome b2. Biochemistry.46.7844-7851.   [Abstract]

Sobrado,P., Kyle, K., Kaul, S., Marwah, A., Arabshahi, I., Turco, M. and Fox, B.G. (2006) Identification of the Binding Region of the [2Fe-2S] Ferredoxin in Stearoyl Acyl Carrier Protein Desaturase: Insight in to the Catalytic complex and Mechanism of Action. Biochemistry.45.4848-4858.   [Abstract]

Sobrado, P., Jedliki, A., Bustos, V.H., Allende, C. C. and Allende, E.J. (2005) The Basic Region of Residues 228-231 of Protein Kinase CK1 is Involved in its Interaction with Axin: Binding to axin does not affect kinase activity. J .Cell. Biochem. 94. 217-224.    [Abstract]

Sobrado, P. and Fitzpatrick, P.F. (2003) Solvent and Primary Deuterium Isotope Effects Show that Lactate CH and OH Bond Cleavage Are Concerted in Y254F Flavocytochrome b2, Consistent With a Hydride Transfer Mechanism. Biochemistry 42.15208-15214.    [Abstract]

Sobrado, P. and Fitzpatrick, P.F. (2003) Analysis of the Role of the Active Site Residue Arg98 in the Flavoprotein Tryptophan 2-Monooxygenase, a Member of the L-Amino Oxidase Family. Biochemistry. 42. 13833-13838.   [Abstract]

Sobrado, P. and Fitzpatrick P. F. (2002) Analysis of the Roles of Amino Acid Residues in the Flavoprotein Tryptophan 2-Monooxygenase Modified by 2-Oxo-3-pentynoate: Characterization of His338, Cys339 and C511 Mutant Enzymes. Archives of Biochemistry and Biophysics. 402. 24-30.    [Abstract]

Sobrado, P., Daubner, S.C. and Fitzpatrick, P.F. (2001) Probing the Relative Timing of Hydrogen Abstraction in the Flavocytochrome b2 Reaction with Primary and Solvent Deuterium Isotope Effects and Mutant Enzymes. Biochemistry. 40. 994-1001    [Abstract]