The objective of this work is to investigate the structure, mechanism of action, and inhibition of three different types of metalloprotein, all of which utilize [4Fe-4S] clusters in catalysis. The reactions catalyzed are reductive dehydroxylations, dehydrations and isomerizations, and all of the proteins are of long-term interest because they are essential for the survival of many pathogens and are not used by humans. The proteins are IspG and IspH, involved in reductive dehydroxylations in isoprenoid biosynthesis;quinolinate synthase (NadA), involved in NAD biosynthesis;and dihydroxyacid dehydratase (DHAD) and isopropylmalate isomerase (LeuCD), involved in amino-acid biosynthesis.
In Aim 1 the first objective is to investigate a previously uncharacterized class of IspHs that are found in many anaerobic bacteria, such as those which cause tetanus and botulism, as well as many which are found in the human microbiome. These proteins are 2-3x larger than most bacterial IspHs and contain an IspH-RPS1 (ribosomal binding protein S1) fusion and might act as iron or oxygen sensors. The second objective is to probe the mechanisms of action of both IspG and IspH by using rapid freeze-quench EPR, calorimetry, site-directed mutagenesis, M?ssbauer, X-ray crystallography, and DFT to characterize reaction intermediates. The third objective is to determine the structures of the 3-domain IspGs and develop novel IspG inhibitors.
Aim 2 involves investigation of NadA, an enzyme whose apo-structure resembles that of IspH but whose structure with its 4Fe-4S cluster is unknown. The objective is to determine its structure and mechanism of action and to develop inhibitors as drug leads (against the organism that causes gastric ulcers and carcinoma).
The third Aim i nvolves two [4Fe-4S] hydratase/isomerases (DHAD and LeuCD). Both are predicted to contain two domains and it appears that there are structural similarities between DHAD, LeuCD, aconitase and fumarase A, a hypothesis we will test. Inhibitors have been reported as herbicides, blocking leucine and other branched chain amino-acid biosynthesis, an effect that with the LeuCD inhibitor is reversed with L- leucine, and M. tuberculosis leuCD knockouts are being developed as TB vaccines, making LeuCD and DHAD potential new drug targets and here, we will develop inhibitors, active in cells, that bind to their [4Fe-4S] clusters.

Public Health Relevance

The project is aimed at understanding the structure and function of a series of metallo-proteins that contain [4Fe-4S] clusters. These proteins are essential for the growth and survival of the organisms that cause tuberculosis, stomach ulcers and malaria but are not used by humans, making them of particular interest as drug targets.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM065307-13
Application #
8758404
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
2002-06-01
Project End
2018-08-31
Budget Start
2014-09-10
Budget End
2015-08-31
Support Year
13
Fiscal Year
2014
Total Cost
$356,850
Indirect Cost
$131,850
Name
University of Illinois Urbana-Champaign
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
041544081
City
Champaign
State
IL
Country
United States
Zip Code
61820
Liu, Meixia; Chen, Chun-Chi; Chen, Lu et al. (2016) Structure and Function of a ""Head-to-Middle"" Prenyltransferase: Lavandulyl Diphosphate Synthase. Angew Chem Int Ed Engl 55:4721-4
Rao, Guodong; Oldfield, Eric (2016) Structure and Function of Four Classes of the 4Fe-4S Protein, IspH. Biochemistry 55:4119-29
Desai, Janish; Wang, Yang; Wang, Ke et al. (2016) Isoprenoid Biosynthesis Inhibitors Targeting Bacterial Cell Growth. ChemMedChem 11:2205-2215
Wang, Yang; Desai, Janish; Zhang, Yonghui et al. (2016) Bacterial Cell Growth Inhibitors Targeting Undecaprenyl Diphosphate Synthase and Undecaprenyl Diphosphate Phosphatase. ChemMedChem 11:2311-2319
Li, Jikun; Feng, Xinxin; Zhu, Wei et al. (2016) Chemical Exchange Saturation Transfer (CEST) Agents: Quantum Chemistry and MRI. Chemistry 22:264-71
Zhang, Lilan; Chen, Chun-Chi; Ko, Tzu-Ping et al. (2016) Moenomycin Biosynthesis: Structure and Mechanism of Action of the Prenyltransferase MoeN5. Angew Chem Int Ed Engl 55:4716-20
García-García, Verónica; Oldfield, Eric; Benaim, Gustavo (2016) Inhibition of Leishmania mexicana Growth by the Tuberculosis Drug SQ109. Antimicrob Agents Chemother 60:6386-9
Desai, Janish; Liu, Yi-Liang; Wei, Hongli et al. (2016) Structure, Function, and Inhibition of Staphylococcus aureus Heptaprenyl Diphosphate Synthase. ChemMedChem 11:1915-23
Kim, Meekyum Olivia; Feng, Xinxin; Feixas, Ferran et al. (2015) A Molecular Dynamics Investigation of Mycobacterium tuberculosis Prenyl Synthases: Conformational Flexibility and Implications for Computer-aided Drug Discovery. Chem Biol Drug Des 85:756-69
Yang, Gyongseon; Zhu, Wei; Wang, Yang et al. (2015) In Vitro and in Vivo Activity of Multitarget Inhibitors against Trypanosoma brucei. ACS Infect Dis 1:388-98

Showing the most recent 10 out of 87 publications