There are three primary goals in this proposal. The first is to understand the molecular details of how the CO sensing transcription factor CooA functions. The binding of CO to the heme iron results in very large structural changes some 20E from the CO. These structural changes switch CooA to the "on-state" such that it specifically recognizes 5'DNA regulatory sequences where CooA promotes specific gene transcription involved in CO oxidation. CooA is a paradigm for how small gaseous ligands lead to large allosteric transitions coupled to important signal transduction processes. In addition, CooA exhibits exquisite ligand selectivity such that only CO results in the allosteric transition and not other biological diatomic gases such as NO and O2. Given that gas sensing now is recognized as a critically important biological process throughout the biosphere, uncovering the structural details of ligand selectivity in CooA is especially important. A second goal is to understand the structural details of heme transport in pathogenic bacteria. Such pathogens must acquire nutrients from the host and one such nutrient is iron. Pathogenic bacteria have developed a complex system where host heme is transported into the cytosol where it is degraded thus releasing iron. The goal of this aim is to determine the structures of all protein components including specific protein complexes. Given that these proteins are unique to bacterial pathogens, they can potentially provide useful therapeutic targets. The third goal is to complete an enzyme engineering study on substrate specificity in peoxidases. Specially, to see if it is possible to engineer in small substrate (such as ascorbate) binding sites into cytochrome c peroxidase which normally uses cytochrome c as a reducing substrate. Relevant to this goal is a newly discovered peroxidase from Leishmania major which oxidizes both ascorbate and cytochrome c and thus appears to be a functional hybrid of cytochrome c and ascorbate peroxidases. The goal here is solve the crystal structure and functionally characterize the enzyme both enzymologically and spectroscopically.

Public Health Relevance

One of the main goals of this proposal centers on the structural biology of iron acquisition from humans by pathogenic bacteria acquire iron from the human host. This will provide the structural basis for designing novel therapeutic agents designed to block iron acquisition by these pathogens and thus prevent infection.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-BCMB-B (02))
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Anderson, Vernon
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University of California Irvine
Schools of Arts and Sciences
United States
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Poulos, Thomas L (2014) Heme enzyme structure and function. Chem Rev 114:3919-62
Tripathi, Sarvind; O'Neill, Maura J; Wilks, Angela et al. (2013) Crystal structure of the Pseudomonas aeruginosa cytoplasmic heme binding protein, Apo-PhuS. J Inorg Biochem 128:131-6
Benabbas, Abdelkrim; Karunakaran, Venugopal; Youn, Hwan et al. (2012) Effect of DNA binding on geminate CO recombination kinetics in CO-sensing transcription factor CooA. J Biol Chem 287:21729-40
Jasion, Victoria S; Poulos, Thomas L (2012) Leishmania major peroxidase is a cytochrome c peroxidase. Biochemistry 51:2453-60
Jasion, Victoria S; Polanco, Julio A; Meharenna, Yergalem T et al. (2011) Crystal structure of Leishmania major peroxidase and characterization of the compound i tryptophan radical. J Biol Chem 286:24608-15
Meharenna, Yergalem T; Doukov, Tzanko; Li, Huiying et al. (2010) Crystallographic and single-crystal spectral analysis of the peroxidase ferryl intermediate. Biochemistry 49:2984-6
Sundaramoorthy, Munirathinam; Gold, Michael H; Poulos, Thomas L (2010) Ultrahigh (0.93A) resolution structure of manganese peroxidase from Phanerochaete chrysosporium: implications for the catalytic mechanism. J Inorg Biochem 104:683-90
Poulos, Thomas L (2010) Thirty years of heme peroxidase structural biology. Arch Biochem Biophys 500:3-12
Ioanoviciu, Alexandra; Meharenna, Yergalem T; Poulos, Thomas L et al. (2009) DevS oxy complex stability identifies this heme protein as a gas sensor in Mycobacterium tuberculosis dormancy. Biochemistry 48:5839-48
Meharenna, Yergalem T; Oertel, Patricia; Bhaskar, B et al. (2008) Engineering ascorbate peroxidase activity into cytochrome c peroxidase. Biochemistry 47:10324-32

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