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 #
5R01GM046436-09
Application #
6386266
Study Section
Metallobiochemistry Study Section (BMT)
Program Officer
Ikeda, Richard A
Project Start
1991-07-01
Project End
2004-03-31
Budget Start
2001-04-01
Budget End
2002-03-31
Support Year
9
Fiscal Year
2001
Total Cost
$273,381
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
Getun, Irina V; Brown, C Kent; Tulla-Puche, Judit et al. (2008) Partially folded bovine pancreatic trypsin inhibitor analogues attain fully native structures when co-crystallized with S195A rat trypsin. J Mol Biol 375:812-23
Valley, Michael P; Brown, C Kent; Burk, David L et al. (2005) Roles of the equatorial tyrosyl iron ligand of protocatechuate 3,4-dioxygenase in catalysis. Biochemistry 44:11024-39
Earhart, Cathleen A; Vetting, Matthew W; Gosu, Ramachandraiah et al. (2005) Structure of catechol 1,2-dioxygenase from Pseudomonas arvilla. Biochem Biophys Res Commun 338:198-205
Vetting, Matthew W; Wackett, Lawrence P; Que Jr, Lawrence et al. (2004) Crystallographic comparison of manganese- and iron-dependent homoprotocatechuate 2,3-dioxygenases. J Bacteriol 186:1945-58
Weinreich, M; Liang, C; Chen, H H et al. (2001) Binding of cyclin-dependent kinases to ORC and Cdc6p regulates the chromosome replication cycle. Proc Natl Acad Sci U S A 98:11211-7
Vetting, M W; Ohlendorf, D H (2000) The 1.8 A crystal structure of catechol 1,2-dioxygenase reveals a novel hydrophobic helical zipper as a subunit linker. Structure 8:429-40
Vetting, M W; D'Argenio, D A; Ornston, L N et al. (2000) Structure of Acinetobacter strain ADP1 protocatechuate 3, 4-dioxygenase at 2.2 A resolution: implications for the mechanism of an intradiol dioxygenase. Biochemistry 39:7943-55
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
Elgren, T E; Orville, A M; Kelly, K A et al. (1997) Crystal structure and resonance Raman studies of protocatechuate 3,4-dioxygenase complexed with 3,4-dihydroxyphenylacetate. Biochemistry 36:11504-13

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