The long term goal of this research is to apply techniques from protein chemistry, molecular biology, and x-ray crystallography to study thoroughly the mechanism, structure, and evolution of the enzymes in two meta-fission pathways-the catechol and homoprotocatechuate meta- fission pathways-the catechol and homoprotocatechuate meta-fission pathways. Such studies have implications for the understanding for the understanding of basic enzyme mechanisms, enol and dienol chemistry , and the evolution of enzyme specificity and catabolic pathways. In addition to addressing these intellectual questions, these studies may assist bio-remediation efforts. During this funding period, we will continue our studies on 4- oxalocrotonate tautomerase (4-OD), 4-oxalocrotonate decarboxylase (4- OD) and vinylpyruvate hydratase (VPH), three sequential enzymes found in the catechol metafission pathway. The proposed studies will address key mechanistic and evolutionary question raised by what appears to be a common in the catechol meta-fission pathway. The proposed studies will address key mechanistic and evolutionary pathways raised by what appears to be a common strategy used by the various meta-fission pathways to degrade catecholic compounds. Our major specific aims, listed in the order of priority, during this funding period will be to: (1) synthesize potential dicarboxylated inhibitors of 4- OT as ligands for crystal structures; (2) delineate the roles of the active site arginines in 4-OT using coded and non-coded amino acids; (3) delineate additional factors governing the reactivity of the catalytic base in 4-OT; (4) determine the 13C kinetic isotope effects on the non- enzymatic and enzymatic decarboxylation of 2-oxo-3-hexenedionate as a function of pH and medium; (5) investigate whether substrate channeling is involved in the 4-ODP/VPH complex; (6) synthesize and evaluate potential inhibitors of VPH; and (7) pursue crystallographic studies of 4- OD/VPH and OHED hydratase. These studies will address long-standing questions about these enzymes as well as new questions raised in the previous funding period. In the process, they will address major evolutionary questions concerning the enzymes of the catechol metafission pathway. The results from these studies serve as a basis for comparison to t he enzyme of other meta- fission pathways as well as the foundation for future experiments and ultimately the genetic manipulations of specificity and activity.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM041239-12
Application #
6476497
Study Section
Bio-Organic and Natural Products Chemistry Study Section (BNP)
Program Officer
Jones, Warren
Project Start
1989-07-01
Project End
2003-11-30
Budget Start
2001-12-01
Budget End
2002-11-30
Support Year
12
Fiscal Year
2002
Total Cost
$253,924
Indirect Cost
Name
University of Texas Austin
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
City
Austin
State
TX
Country
United States
Zip Code
78712
LeVieux, Jake A; Baas, Bert-Jan; Kaoud, Tamer S et al. (2017) Kinetic and structural characterization of a cis-3-Chloroacrylic acid dehalogenase homologue in Pseudomonas sp. UW4: A potential step between subgroups in the tautomerase superfamily. Arch Biochem Biophys 636:50-56
Johnson Jr, William H; Stack, Tyler M M; Taylor, Stephanie M et al. (2016) Stereochemical Consequences of Vinylpyruvate Hydratase-Catalyzed Reactions. Biochemistry 55:4055-64
Guimarães, Samuel L; Coitinho, Juliana B; Costa, Débora M A et al. (2016) Crystal Structures of Apo and Liganded 4-Oxalocrotonate Decarboxylase Uncover a Structural Basis for the Metal-Assisted Decarboxylation of a Vinylogous ?-Keto Acid. Biochemistry 55:2632-45
Araújo, Simara Semíramis de; Neves, Cíntia Mara Leal; Guimarães, Samuel Leite et al. (2015) Structural and kinetic characterization of recombinant 2-hydroxymuconate semialdehyde dehydrogenase from Pseudomonas putida G7. Arch Biochem Biophys 579:8-17
Huddleston, Jamison P; Burks, Elizabeth A; Whitman, Christian P (2014) Identification and characterization of new family members in the tautomerase superfamily: analysis and implications. Arch Biochem Biophys 564:189-96
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
Burks, Elizabeth A; Yan, Wupeng; Johnson Jr, William H et al. (2011) Kinetic, crystallographic, and mechanistic characterization of TomN: elucidation of a function for a 4-oxalocrotonate tautomerase homologue in the tomaymycin biosynthetic pathway. Biochemistry 50:7600-11
Srivastava, Dhiraj; Zhu, Weidong; Johnson Jr, William H et al. (2010) The structure of the proline utilization a proline dehydrogenase domain inactivated by N-propargylglycine provides insight into conformational changes induced by substrate binding and flavin reduction. Biochemistry 49:560-9
Burks, Elizabeth A; Fleming, Christopher D; Mesecar, Andrew D et al. (2010) Kinetic and structural characterization of a heterohexamer 4-oxalocrotonate tautomerase from Chloroflexus aurantiacus J-10-fl: implications for functional and structural diversity in the tautomerase superfamily . Biochemistry 49:5016-27
Almrud, Jeffrey J; Dasgupta, Rakhi; Czerwinski, Robert M et al. (2010) Kinetic and structural characterization of DmpI from Helicobacter pylori and Archaeoglobus fulgidus, two 4-oxalocrotonate tautomerase family members. Bioorg Chem 38:252-9

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