In order to advance our understanding of the chemistry and biology of the greater family of manganese- requiring enzymes, we propose to explore structure-function relationships in human arginase I as well as the arginase-related metalloenzymes histone deacetylase 8 and polyamine deacetylase. Human arginase I contains a binuclear manganese cluster required for the hydrolysis of L-arginine to form L-ornithine and urea, and our studies indicate that catalysis proceeds through a mechanism in which both metal ions function to activate a metal-bridging hydroxide ion as the catalytic nucleophile. We have determined the structure of this enzyme to 1.29 ? resolution, and we will use this structure to guide the design of inhibitors and biosensors. Recent discoveries show that arginase is upregulated in various diseases such as atherosclerosis, asthma, and cancer, so our studies will expand the repertoire of chemical compounds that will potentially be useful for the treatment and diagnosis of human disease. Given the newly-discovered and unexpected structural relationship between the arginases and metal- dependent deacetylases, our structural and functional studies will illuminate important mechanistic parallels between these enzyme families. Intriguingly, the Zn2+ site of the deacetylase corresponds to the Mn2+B site of arginase, but the deacetylase does not contain a metal binding site corresponding to Mn2+A of arginase. Thus, the stoichiometry of metal binding has diverged in the evolution of the arginases and the deacetylases from a common metalloenzyme precursor. Intriguingly, the biologically preferred metal ion of human histone deacetylase-8 is believed to be Fe2+. Therefore, we will determine the structures of the Fe2+-substituted enzyme, its site-specific variants, and its substrate and inhibitor complexes. Since this enzyme is a validated drug target for cancer chemotherapy, it is important to thoroughly understand structure-function relationships in the form of the metalloenzyme that is found in vivo. Overall, the proposed research will provide a greater structural and functional understanding of metal ion specificity (Mn2+, Zn2+, Fe2+) and stoichiometry in the evolution of the arginases and the arginase-related deacetylases.

Public Health Relevance

Structural and functional studies of human arginase I, human histone deacetylase-8, and bacterial polyamine deacetylase will facilitate the design of potential new drugs that can be used to treat atherosclerosis, asthma, and cancer. Additionally, our studies will enable the design and development of biosensors that may be useful in the early diagnosis of human disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM049758-18
Application #
8212361
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Smith, Ward
Project Start
1994-05-01
Project End
2014-01-31
Budget Start
2012-02-01
Budget End
2013-01-31
Support Year
18
Fiscal Year
2012
Total Cost
$313,680
Indirect Cost
$110,812
Name
University of Pennsylvania
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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Ilies, Monica; Dowling, Daniel P; Lombardi, Patrick M et al. (2011) Synthesis of a new trifluoromethylketone analogue of l-arginine and contrasting inhibitory activity against human arginase I and histone deacetylase 8. Bioorg Med Chem Lett 21:5854-8
Ilies, Monica; Di Costanzo, Luigi; Dowling, Daniel P et al. (2011) Binding of ?,?-disubstituted amino acids to arginase suggests new avenues for inhibitor design. J Med Chem 54:5432-43
D'Antonio, Edward L; Christianson, David W (2011) Crystal structures of complexes with cobalt-reconstituted human arginase I. Biochemistry 50:8018-27
Lombardi, Patrick M; Angell, Heather D; Whittington, Douglas A et al. (2011) Structure of prokaryotic polyamine deacetylase reveals evolutionary functional relationships with eukaryotic histone deacetylases. Biochemistry 50:1808-17

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