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Biochemistry of oxygenases: Mechanistic studies of a cofactor-independent, CO-formingm dioxygenase belonging to the a/ß-hydrolase fold family

Subject Area Biophysics
Term from 2008 to 2014
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 91767900
 
Final Report Year 2014

Final Report Abstract

Chemistry and biology always face the difficulty that reactions of organic compounds with dioxygen are restricted by the rule of spin conservation. Molecular oxygen in its electronic ground state is a triplet state biradical, which makes O2 chemically inert toward direct reactions with singlet state organic molecules. Hence, to overcome this restriction, activation of either the organic molecule or O2 is required. The majority of enzymes that use dioxygen for catalysis depend on transition metal ions or organic cofactors. However, a group of O2-metabolizing enzymes overcome the restriction without requirement for a metal ion cofactor or an organic cofactor. The cofactor-independent enzyme 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) is involved in degradation of 2-methylquinoline by Arthrobacter sp. Rue61a. Hod possesses a classical α/βhydrolase fold core domain, with an additional cap domain, and a structurally conserved Ser-His-Asp triad in its active site. In the hydrolase members of the family, this so-called “catalytic triad” is required for catalysis. The catalytic action of the “true” α/β-hydrolases usually relies mainly on the nucleophilicity of the triad’s serine. However, in Hod, the active-site serine is not essential for the physiological reaction, i.e., the dioxygenolytic ring cleavage of 1H-3-hydroxy-4-oxoquinaldine to carbon monoxide and N-acetylanthranilate. Nevertheless, Hod possesses hydrolase-like properties, and the active-site serine is able to act as a nucleophile: Inactivation of Hod by isatoic anhydride, which is a suicide substrate for some serine proteases, is based on the classical serine hydrolase mechanism, involving nucleophilic attack of the triad’s serine at the carbonyl group of isatoic anhydride, with subsequent ester bond cleavage. Decarboxylation of this acyl-enzyme intermediate releases CO2 and yields a stable covalent anthraniloyl-serine intermediate. Recent data showing very slow release of anthranilate, accompanied by reactivation of Hod, indicate a hydrolytic cleavage of the anthraniloyl-enzyme and thereby suggest catalytic promiscuity of Hod. In the dioxygenase reaction catalyzed by Hod, the first step is the deprotonation of the 3-OH group of the substrate 1H-3-hydroxy-4-oxoquinaldine to form an enzyme-bound anion. This deprotonation is mediated by the active-site histidine, which together with the conserved Asp forms a charge relay system. Thus, whereas Hod-mediated hydrolysis of isatoic anhydride follows a nucleophilic, serine hydrolase like mechanism, the physiological reaction of Hod involves on a non-nucleophilic generalbase mechanism. To further elucidate the mechanism of cofactor-independent dioxygenolysis, we characterized the mode of O2 activation in the reaction pathway of Hod, and identified catalytic intermediates. Chemical analysis and electron paramagnetic resonance spectroscopic data revealed that O2 activation in the enzyme’s active site relies on single electron transfer from the bound substrate anion to O2 to form a [substrate radical – superoxide radical] pair, which recombines to a C2-hydroperoxide intermediate. This is the first time that experimental evidence was provided for radical pair formation by a cofactor-less oxygenase. In this reaction pathway, a major role of Hod, besides activating the substrate by general base catalysis and stabilizing transition states and reactive intermediates along the reaction pathway, is to provide an environment that allows electron transfer from the substrate anion to O2. Since the enzyme utilizes the induced intrinsic reactivity of its enzymebound substrate anion for O2 activation, it is a “substrate-assisted oxygenase”.

Publications

  • (2010) Cofactor-independent oxidases and oxygenases. Appl. Microbiol. Biotechnol. 86: 791-804
    Fetzner S, Steiner RA
  • (2010) Structural basis for cofactorindependent dioxygenation of N-heteroaromatic compounds at the α/β hydrolase fold. Proc. Natl. Acad. Sci. USA 107: 657-662
    Steiner RA, Janßen HJ, Roversi P, Oakley AJ, Fetzner S
  • (2011) Redox regulation of calcium ion channels: Chemical and physiological aspects, Cell Calcium 50: 407-423
    Bogeski, I., Kappl, R., Kummerow, C., Gulaboski, R., Hoth, M., and Niemeyer, B. A.
  • (2012) Hydrolase-like properties of a cofactor-independent dioxygenase. ChemBioChem 13: 1125- 1127
    Thierbach S, Büldt-Karentzopoulos K, Dreiling A, Hennecke U, König S, Fetzner S
    (See online at https://doi.org/10.1002/cbic.201200152)
  • (2012) Ring-cleaving dioxygenases with a cupin fold. Appl. Environ. Microbiol. 78: 2505-2514
    Fetzner S
  • (2013) Hydroxylated derivatives of dimethoxy-1,4-benzoquinone as redox switchable earth-alkaline metal ligands and radical scavengers, Scientific Reports 3:1865
    Gulaboski, R., Bogeski, I., Mirceski, V., Saul, S., Pasieka, B., Haeri, H. H., Stefova, M., Stanoeva, J. P., Mitrev, S., Hoth, M., and Kappl, R
    (See online at https://doi.org/10.1038/srep01865)
  • (2014) Substrate-assisted O2 activation in a cofactor-independent dioxygenase. Chem. Biol. 21: 217-225. Referred to by: Bugg, T.D.H.: How to break the rules of dioxygen activation. Chem. Biol. 21: 168-169
    Thierbach S, Bui N, Zapp J, Chhabra SR, Kappl R, Fetzner S
    (See online at https://doi.org/10.1016/j.chembiol.2013.11.013)
 
 

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