Research supported by this grant in recent years has focused on what we have defined as the non-heme iron reductive paradigm for combating oxidative and nitrosative stress. This novel paradigm involves reductive scavenging of toxic reduced oxygen and nitrogen species by a group of bacterial and archaeal non-heme iron enzymes. Genetic evidence associates the functions of these enzymes in numerous bacterial and archaeal species. Based on genome sequences, these enzymes are found predominantly in air-sensitive bacteria, including those that constitute the majority of the normal human gut flora as well as human pathogens. These enzymes may protect against the oxidative and nitrosative burst of macrophages, which is the human host's initial response to infection. In the present renewal proposal, one specific aim focuses on the mechanism of reductive nitric oxide scavenging by a novel enzyme containing a combination of flavin and diiron cofactors at its active site. Single turnover experiments will be used to identify the Fe-NO species formed upon reactions of the diiron sites with nitric oxide and to determine their catalytic relevance. These themes are then logically extended to bacterial O2 sensing and signaling, induction of biofilm formation and virulence, all involving bacterial non-heme iron proteins. Enzymes from Vibrio cholerae, the bacterium causing epidemic cholerae, catalyzing formation and decay of the bacterial "second messenger", cyclic-di-guanosine monophosphate, are the focus in these latter two specific aims. These enzymes are widespread in bacteria but have not been found in higher eukaryotes. Inhibitors of these enzymes could, therefore, constitute new classes of antibiotics. Intelligent design of antibiotics requires an understanding of their catalytic and/or sensing/signaling mechanisms. These mechanisms are the overall goal of this research.

Public Health Relevance

The enzymes proposed for study may provide protection to pathogenic bacteria against the oxidative and nitrosative burst of macrophages, which is the human host's initial response to infection. Sensing/signaling proteins from Vibrio cholerae, the bacterium causing epidemic cholera, are prominently featured in the proposed research. When the structure and function of these enzymes is understood, inhibitors of these enzymes could be designed as new classes of antibiotics.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM040388-22
Application #
8471112
Study Section
Special Emphasis Panel (ZRG1-BCMB-B (03))
Program Officer
Anderson, Vernon
Project Start
1988-07-01
Project End
2015-05-31
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
22
Fiscal Year
2013
Total Cost
$320,424
Indirect Cost
$93,649
Name
University of Texas Health Science Center San Antonio
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
800189185
City
San Antonio
State
TX
Country
United States
Zip Code
78249
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Okamoto, Yasunori; Onoda, Akira; Sugimoto, Hiroshi et al. (2014) H2O2-dependent substrate oxidation by an engineered diiron site in a bacterial hemerythrin. Chem Commun (Camb) 50:3421-3
Caranto, Jonathan D; Weitz, Andrew; Hendrich, Michael P et al. (2014) The nitric oxide reductase mechanism of a flavo-diiron protein: identification of active-site intermediates and products. J Am Chem Soc 136:7981-92
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Hayashi, Takahiro; Caranto, Jonathan D; Wampler, David A et al. (2010) Insights into the nitric oxide reductase mechanism of flavodiiron proteins from a flavin-free enzyme. Biochemistry 49:7040-9
Hillmann, Falk; Riebe, Oliver; Fischer, Ralf-Jorg et al. (2009) Reductive dioxygen scavenging by flavo-diiron proteins of Clostridium acetobutylicum. FEBS Lett 583:241-5

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