Dioxygenases catalyze the incorporation of both atoms of molecular oxygen into a substrate. In bacteria, this ring- opening reaction is a key step in the degradation pathway for many aromatic compounds found in the environment. In plants and animals, dioxygenases are involved in the metabolism of indoles, aromatic amino acids, arachidonic acids and prostaglandins. Dioxygenases are typically metalloproteins many of which require a non-heme mononuclear iron center as a cofactor. This project has been understaken to discover the structural foundations for catalysis in these metalloenzymes. The principal target of this project has been protocatechuate 3,4-dioxygenase (3,4-PCD; Fe+3 cofactor, cleaves aromatic rings between hydroxyls) which has been used as a model system. To date for 3,4-PCD from Pseudomonas putida we have determined the refined structures of the wild-type enzyme, of 6 mutants and of 16 substrate/inhibitor complexes; for 3,4-PCD from Acinetobacter calcoaceticus we have determined the refined structures of the wild-type enzyme, of a mutant, and of 4 complexes; for 3,4-PCD from Brevibacterium fuscum we have solved the structure of the wild-type enzyme. Other structures solved include catechol 1,2- dioxygenase (1,2-CTD; Fe+3 cofactor, cleaves aromatic rings between hydroxyls) from Pseudomonas arvilla and A. calcoaceticus and homoprotocatechuate 2,3-dioxygenase (2,3-HPCD; cleaves aromatic rings adjacent to hydroxyls) from B. fuscum (Fe+2 cofactor) and from Arthrobacter globiformis CM-2 (Mn+2 cofactor). This project builds upon a wealth of spectroscopic, kinetic and genetic data gathered over the past 35 years in a number of laboratories key among which are those of our collaborators. Thus our expertise in structural analysis and mutagenesis synergizes with those of our collaborators in spectroscopy, kinetics and genetics to produce a coordinated analysis of this family of metalloenzymes. Questions to be addressed by this combined approach include: What is the difference between Fe+3, Fe+2 and Mn+2 dioxygenases? How does metal ligation change during catalysis or as a function of oxidation state? What is the role of the active site residues in binding, positioning, and preparing metal, substrate and oxygen for catalysis? What is the basis of substrate specificity? And, what is the basis for selecting between intradiol and extradiol cleavage?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM046436-10
Application #
6519459
Study Section
Metallobiochemistry Study Section (BMT)
Program Officer
Ikeda, Richard A
Project Start
1991-07-01
Project End
2004-03-31
Budget Start
2002-04-01
Budget End
2003-03-31
Support Year
10
Fiscal Year
2002
Total Cost
$281,345
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Biochemistry
Type
Schools of Medicine
DUNS #
168559177
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
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D'Argenio, D A; Vetting, M W; Ohlendorf, D H et al. (1999) Substitution, insertion, deletion, suppression, and altered substrate specificity in functional protocatechuate 3,4-dioxygenases. J Bacteriol 181:6478-87
Frazee, R W; Orville, A M; Dolbeare, K B et al. (1998) The axial tyrosinate Fe3+ ligand in protocatechuate 3,4-dioxygenase influences substrate binding and product release: evidence for new reaction cycle intermediates. Biochemistry 37:2131-44
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