Research Article: Purification and characterization of hydroquinone dioxygenase from Sphingomonas sp. strain TTNP3

Date Published: May 27, 2011

Publisher: Springer

Author(s): Boris A Kolvenbach, Markus Lenz, Dirk Benndorf, Erdmann Rapp, Jan Fousek, Cestmir Vlcek, Andreas Schäffer, Frédéric LP Gabriel, Hans-Peter E Kohler, Philippe FX Corvini.


Hydroquinone-1,2-dioxygenase, an enzyme involved in the degradation of alkylphenols in Sphingomonas sp. strain TTNP3 was purified to apparent homogeneity. The extradiol dioxygenase catalyzed the ring fission of hydroquinone to 4-hydroxymuconic semialdehyde and the degradation of chlorinated and several alkylated hydroquinones. The activity of 1 mg of the purified enzyme with unsubstituted hydroquinone was 6.1 μmol per minute, the apparent Km 2.2 μM. ICP-MS analysis revealed an iron content of 1.4 moles per mole enzyme. The enzyme lost activity upon exposure to oxygen, but could be reactivated by Fe(II) in presence of ascorbate. SDS-PAGE analysis of the purified enzyme yielded two bands of an apparent size of 38 kDa and 19 kDa, respectively. Data from MALDI-TOF analyses of peptides of the respective bands matched with the deduced amino acid sequences of two neighboring open reading frames found in genomic DNA of Sphingomonas sp strain TTNP3. The deduced amino acid sequences showed 62% and 47% identity to the large and small subunit of hydroquinone dioxygenase from Pseudomonas fluorescens strain ACB, respectively. This heterotetrameric enzyme is the first of its kind found in a strain of the genus Sphingomonas sensu latu.

Partial Text

Both Sphingomonas sp. strain TTNP3 and Sphingobium xenophagum Bayram are able to degrade several branched isomers of nonylphenol and bisphenol A, well-known endocrine disruptors, by ipso substitution. i.e. ipso-hydroxylation and subsequent detachment of the side chain of the alkylphenol. In these pathways hydroquinone is formed as a key metabolite (Kolvenbach et al. 2007; Corvini et al. 2006; Gabriel et al. 2007a; Gabriel et al. 2007b; Gabriel et al. 2005). Hydroquinone (HQ) is also a key intermediate in the degradation of several other compounds of environmental importance, such as 4-nitrophenol (Spain and Gibson 1991), γ-hexachlorocyclohexane (Miyauchi et al. 1999), 4-hydroxyacetophenone (Moonen et al. 2008a) and 4-aminophenol (Takenaka et al. 2003).

We were able to isolate and characterize a protein from Sphingomonas sp. strain TTNP3 that catalyzes the Fe2+- and O2-dependent conversion of HQ to 4-hydroxymuconic semialdehyde. Like nonylphenol ipso-hydroxylases, i.e. the first enzyme in the degradation pathway of nonylphenol and bisphenol A (Kolvenbach et al. 2007; Gabriel et al. 2007a; Gabriel et al. 2007b; Gabriel et al. 2005; Corvini et al. 2006), the HQDO represents an interesting class of enzymes that has been little studied.

The authors declare that they have no competing interests.

BAK carried out the enzyme purification and biochemical experiments and drafted the manuscript. ML performed IPC-MS analyses. DB and ER carried out protein analysis and identification. JF and CV performed the genome sequencing and assembly and provided nucleotide sequence data. FLPG elaborated GC-MS data, conceived fragmentation patterns and commented on the manuscript. HPEK participated in the design of the study and commented on the manuscript. AS and PFXC participated in the design of the study, commented on the manuscript and supervised the Ph.D. thesis of BAK from which large parts of this study originated.




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