Methionine is an important source of alkylating equivalents in living cells. As S-adenosylmethionine (SAM), the terminal methyl group of methionine is used in a wide array of transmethylation reactions in vivo, and the ethylglycine moiety feeds into polyamine biosynthesis in rapidly proliferating cells. Interest in the metabolic fate of methionine is raised by the observation that many human cancer cell lines are methionine dependent, and that methylthioadenosine (MTA), a product of SAM metabolism, is a potent feedback inhibitor of polyamine biosynthesis, which in turn stops DNA replication and prevents continuation of the cell cycle. There is evidence suggesting that the methionine salvage pathway, which is responsible for recycling the S-OH3 group from MTA to methionine, is defective in methionine-dependent cancer cell lines. This proposal describes an in-depth investigation into the structure and function of a remarkable metalloenzyme from the methionine salvage pathway of the bacterium Klebsiella pneumoniae, acireductone dioxygenase (ARD). ARD can catalyze two different reactions with the same substrate depending upon the metal ion that is bound to the enzyme active site. ARD containing a single Ni+2 ion catalyzes the oxidative cleavage of acireductone, an advanced intermediate in the methionine salvage pathway, into beta-methylthiopropionate, formate and carbon monoxide (CO) in an off-pathway shunt. If a Fe+2 ion is bound instead of nickel (ARD'), the on-pathway reaction leading to formate and the ketoacid precursor of methionine, alpha-keto-gamma-methylthiobutyrate is catalyzed. A structure for Ni-bound ARD has recently been solved by NMR methods, and mechanistic studies have provided evidence for a radical mechanism for the reaction catalyzed by ARD. The general aims of the proposed work include: (1) Determination of the solution structure of ARD' and an analysis of the structural differences between ARD and ARD' that lead to their different activities. Site-directed mutagenesis, enzyme activity and kinetics measurements and NMR studies of stable enzyme-substrate complexes will be performed. (2) Expression, isolation, purification and characterization of ARD homologues from two eukaryotes, rice (Oriza sativa) and humans (Homo sapiens). In particular, evidence for in-vitro and in-vivo ARD activity will be sought (i.e., CO production), since there is now considerable evidence that CO can act as a signaling molecule and an inhibitor of apoptosis (programmed cell death).

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM067786-03
Application #
6890417
Study Section
Metallobiochemistry Study Section (BMT)
Program Officer
Preusch, Peter C
Project Start
2003-05-05
Project End
2007-04-30
Budget Start
2005-05-01
Budget End
2006-04-30
Support Year
3
Fiscal Year
2005
Total Cost
$209,250
Indirect Cost
Name
Brandeis University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
616845814
City
Waltham
State
MA
Country
United States
Zip Code
02454
Deshpande, Aditi R; Pochapsky, Thomas C; Petsko, Gregory A et al. (2017) Dual chemistry catalyzed by human acireductone dioxygenase. Protein Eng Des Sel 30:197-204
Deshpande, Aditi R; Pochapsky, Thomas C; Ringe, Dagmar (2017) The Metal Drives the Chemistry: Dual Functions of Acireductone Dioxygenase. Chem Rev 117:10474-10501
Deshpande, Aditi R; Wagenpfeil, Karina; Pochapsky, Thomas C et al. (2016) Metal-Dependent Function of a Mammalian Acireductone Dioxygenase. Biochemistry 55:1398-407
Pochapsky, Susan Sondej; Sunshine, Joel C; Pochapsky, Thomas C (2008) Completing the circuit: direct-observe 13C,15N double-quantum spectroscopy permits sequential resonance assignments near a paramagnetic center in acireductone dioxygenase. J Am Chem Soc 130:2156-7
Chai, Sergio C; Ju, Tingting; Dang, Marina et al. (2008) Characterization of metal binding in the active sites of acireductone dioxygenase isoforms from Klebsiella ATCC 8724. Biochemistry 47:2428-38
Oram, Shane W; Ai, Junkui; Pagani, Gina M et al. (2007) Expression and function of the human androgen-responsive gene ADI1 in prostate cancer. Neoplasia 9:643-51
Pochapsky, Thomas C; Pochapsky, Susan S; Ju, Tingting et al. (2006) A refined model for the structure of acireductone dioxygenase from Klebsiella ATCC 8724 incorporating residual dipolar couplings. J Biomol NMR 34:117-27
Ju, Tingting; Goldsmith, Rachel Beaulieu; Chai, Sergio C et al. (2006) One protein, two enzymes revisited: a structural entropy switch interconverts the two isoforms of acireductone dioxygenase. J Mol Biol 363:823-34
OuYang, Bo; Pochapsky, Susan Sondej; Pagani, Gina M et al. (2006) Specific effects of potassium ion binding on wild-type and L358P cytochrome P450cam. Biochemistry 45:14379-88
Sauter, Margret; Lorbiecke, Rene; Ouyang, Bo et al. (2005) The immediate-early ethylene response gene OsARD1 encodes an acireductone dioxygenase involved in recycling of the ethylene precursor S-adenosylmethionine. Plant J 44:718-29

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