Mechanism of taurine: alpha-ketoglutarate dioxygenase

Project: Research project

Description

DESCRIPTION (provided by applicant) The Fe(ll)-/a-ketoglutarate-dependent dioxygenases couple oxidative decarboxylation of a-ketoglutarate with hydroxylation of unactivated carbon atoms on a variety of substrates. Enzymes in this class catalyze important reactions in organisms from bacteria to humans and have essential roles in such critical processes as sensing of oxygen and response to hypoxia in mammals; synthesis of B-lactam antibiotics, collagen, and fatty acids in a range of organisms; and repair of DNA alkylation damage in bacteria and humans. In addition, dysfunction of enzymes in the class of enzymes has been associated with numerous disease states. It has been proposed that these enzymes operate by a common mechanism. Despite extensive investigation in many laboratories, there had been, until our recent work, no direct evidence for any of the several intermediate states proposed to follow after reaction of the enzyme with molecular oxygen. We have recently obtained the first direct evidence for an oxidized iron intermediate in the reaction catalyzed by one enzyme in this class, taurine/a-ketoglutarate dioxygenase (TauD), and have shown that the novel complex accumulates to levels sufficient for a thorough spectroscopic characterization. Preliminary data indicate that the intermediate has a formal Fe(IV) oxidation state and suggest two most likely structures for the complex. The main objectives of this research project are: (1) To place the novel Fe(IV) intermediate in the sequence of events leading to O2 activation, a-ketoglutarate oxidative decarboxylation, and taurine hydroxylation by a combination of rapid kinetics experiments in order to limit possible structural assignments and choose between the two most likely structures; (2) To rigorously characterize this novel reaction intermediate by a combination of spectroscopic techniques in order to elucidate its electronic and geometric structure; and (3) To use modified reactants (substrate analogues and site directed TauD variants) to accumulate and then trap and characterize intermediate states in the reaction pathway that are not accessible in the reaction with the normal components. Achievement of these goals will provide new and unprecedented insight into the molecular details of the catalytic mechanism of TauD and, more importantly, of the Fe(ll)/a-ketoglutarate-dependent dioxygenases in general.
StatusFinished
Effective start/end date1/1/047/31/18

Funding

  • National Institutes of Health: $363,538.00
  • National Institutes of Health: $251,140.00
  • National Institutes of Health: $264,936.00
  • National Institutes of Health: $363,538.00
  • National Institutes of Health: $363,538.00
  • National Institutes of Health: $363,538.00
  • National Institutes of Health: $251,140.00
  • National Institutes of Health: $264,913.00
  • National Institutes of Health: $258,665.00

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Dioxygenases
Enzymes
Taurine
Oxidation
Hydroxylation
Carbon
Chemical activation
Substrates
Anti-Bacterial Agents
Inositol Oxygenase
Bacteria
taurine-alpha-ketoglutarate dioxygenase
Iron
Fosfomycin
Lactams
Reaction intermediates
Atoms
Mammals
Carbapenems
Molecular oxygen