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Journal of Bacteriology, August 2008, p. 5199-5209, Vol. 190, No. 15
0021-9193/08/$08.00+0     doi:10.1128/JB.01945-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Hydroquinone Dioxygenase from Pseudomonas fluorescens ACB: a Novel Member of the Family of Nonheme-Iron(II)-Dependent Dioxygenases{triangledown}

Mariëlle J. H. Moonen,1,{dagger} Silvia A. Synowsky,2,{dagger} Willy A. M. van den Berg,1 Adrie H. Westphal,1 Albert J. R. Heck,2 Robert H. H. van den Heuvel,2,{ddagger} Marco W. Fraaije,3 and Willem J. H. van Berkel1*

Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands,1 Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands,2 Laboratory of Biochemistry, Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands3

Received 14 December 2007/ Accepted 16 May 2008

Hydroquinone 1,2-dioxygenase (HQDO), an enzyme involved in the catabolism of 4-hydroxyacetophenone in Pseudomonas fluorescens ACB, was purified to apparent homogeneity. Ligandation with 4-hydroxybenzoate prevented the enzyme from irreversible inactivation. HQDO was activated by iron(II) ions and catalyzed the ring fission of a wide range of hydroquinones to the corresponding 4-hydroxymuconic semialdehydes. HQDO was inactivated by 2,2'-dipyridyl, o-phenanthroline, and hydrogen peroxide and inhibited by phenolic compounds. The inhibition with 4-hydroxybenzoate (Ki = 14 µM) was competitive with hydroquinone. Online size-exclusion chromatography-mass spectrometry revealed that HQDO is an {alpha}2β2 heterotetramer of 112.4 kDa, which is composed of an {alpha}-subunit of 17.8 kDa and a β-subunit of 38.3 kDa. Each β-subunit binds one molecule of 4-hydroxybenzoate and one iron(II) ion. N-terminal sequencing and peptide mapping and sequencing based on matrix-assisted laser desorption ionization—two-stage time of flight analysis established that the HQDO subunits are encoded by neighboring open reading frames (hapC and hapD) of a gene cluster, implicated to be involved in 4-hydroxyacetophenone degradation. HQDO is a novel member of the family of nonheme-iron(II)-dependent dioxygenases. The enzyme shows insignificant sequence identity with known dioxygenases.

* Corresponding author. Mailing address: Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands. Phone: 31 317 482861. Fax: 31 317 484801. E-mail: willem.vanberkel{at}wur.nl

{triangledown} Published ahead of print on 23 May 2008.

{dagger} M.H.H.M. and S.A.S. contributed equally to the paper.

{ddagger} Present address: NV Organon, Postbus 20, 5340 BH Oss, The Netherlands.

Journal of Bacteriology, August 2008, p. 5199-5209, Vol. 190, No. 15
0021-9193/08/$08.00+0     doi:10.1128/JB.01945-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Zhang, J.-J., Liu, H., Xiao, Y., Zhang, X.-E., Zhou, N.-Y. (2009). Identification and Characterization of Catabolic para-Nitrophenol 4-Monooxygenase and para-Benzoquinone Reductase from Pseudomonas sp. Strain WBC-3. J. Bacteriol. 191: 2703-2710 [Abstract] [Full Text]  
  • Moonen, M. J. H., Kamerbeek, N. M., Westphal, A. H., Boeren, S. A., Janssen, D. B., Fraaije, M. W., van Berkel, W. J. H. (2008). Elucidation of the 4-Hydroxyacetophenone Catabolic Pathway in Pseudomonas fluorescens ACB. J. Bacteriol. 190: 5190-5198 [Abstract] [Full Text]  


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