Studies of microbial biosynthetic and catabolic pathways provide major insights about enzyme mechanism, specificity, structure, regulation, and evolution. Harnessing these insights and the individual enzymes has many practical uses ranging from the synthesis of new drug compounds to bioremediation. This application will address two questions: how the C ring of the antibiotic antitumor agent tomaymycin is made and how polycyclic aromatic hydrocarbons (PAHs) are degraded by one major bacterial pathway. Both questions arose in the course of our studies on 4-oxalocrotonate tautomerase (4-OT) and other tautomerase superfamily members. 4-OT catalyzes a proton transfer reaction in the meta-fission pathway for the degradation of aromatic hydrocarbons. TomN, a newly discovered 4-OT homologue, led us to the tomaymycin pathway because it reportedly catalyzes a proton transfer reaction using a very different substrate. The second question emerged from our extensive use of acetylene compounds as mechanistic probes and inhibitors of 4-OT and other superfamily members. The answer to the first question advances our understanding of the biosynthesis of a large and highly diverse group of natural products, the pyrrolo[1,4]benzodiazepines that are important sources of antimicrobial and anticancer drugs and drug leads. The answer to the second question advances our understanding of PAH degradative pathways, notably ones used for the degradation of higher molecular weight PAHs, which, along with the lower molecular weight PAHs, are highly toxic and persistent environmental contaminants. Using a combination of mechanistic, biochemical, structural, and kinetic techniques, these questions will be addressed in three specific aims.
These aims are to: (1) identify the reactions that convert L-dopa to the C-ring of tomaymycin;(2) identify the key elements of the TomK mechanism;and (3) establish the mechanism and determinants of specificity for the hydratase/aldolases in the naphthalene, phenanthrene, and fluoranthene degradative pathways. The results will also address our long- term goals, which are to obtain a more comprehensive understanding of the relationship between structure and function in enzyme-catalyzed reactions and to delineate the factors that govern protein structure, substrate recognition, and reaction specificity.

Public Health Relevance

The proposed research will determine how Nature makes a crucial part of an anticancer agent known as tomaymycin, and set the stage for generating similar drugs that are more potent, less costly, and less toxic. The research will also determine how bacteria process polycyclic aromatic hydrocarbons, which are persistent and toxic environmental contaminants. The knowledge can assist in the development of bioremediation technologies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM041239-18A1
Application #
8632522
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Barski, Oleg
Project Start
1989-07-01
Project End
2017-11-30
Budget Start
2014-01-01
Budget End
2014-11-30
Support Year
18
Fiscal Year
2014
Total Cost
$269,775
Indirect Cost
$79,775
Name
University of Texas Austin
Department
None
Type
Schools of Pharmacy
DUNS #
170230239
City
Austin
State
TX
Country
United States
Zip Code
78712
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Terrell, Cassidy R; Burks, Elizabeth A; Whitman, Christian P et al. (2013) Structural and kinetic characterization of two 4-oxalocrotonate tautomerases in Methylibium petroleiphilum strain PM1. Arch Biochem Biophys 537:113-24
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