Purification and characterization of a catechol 1,2-dioxygenase from a phenol degrading Candida albicans TL3
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O RI G I NAL PAPE R
PuriWcation and characterization of a catechol 1,2-dioxygenase from a phenol degrading Candida albicans TL3 San-Chin Tsai · Yaw-Kuen Li
Received: 31 May 2006 / Revised: 29 September 2006 / Accepted: 13 October 2006 / Published online: 7 November 2006 © Springer-Verlag 2006
Abstract A eukaryotic catechol 1,2-dioxygenase (1,2CTD) was produced from a Candida albicans TL3 that possesses high tolerance for phenol and strong phenol degrading activity. The 1,2-CTD was puriWed via ammonium sulfate precipitation, Sephadex G-75 gel Wltration, and HiTrap Q Sepharose column chromatography. The enzyme was puriWed to homogeneity and found to be a homodimer with a subunit molecular weight of 32,000. Each subunit contained one iron. The optimal temperature and pH were 25°C and 8.0, respectively. Substrate analysis showed that the puriWed enzyme was a type I catechol 1,2-dioxygenase. This is the Wrst time that a 1,2-CTD from a eukaryote (Candida albicans) has been characterized. Peptide sequencing on fragments of 1,2-CTD by Edman degradation and MALDI-TOF/TOF mass analyses provided information of amino acid sequences for BLAST analysis, the outcome of the BLAST revealed that this eukaryotic 1,2-CTD has high identity with a hypothetical protein, CaO19_12036, from Candida albicans SC5314. We conclude that the hypothetical protein is 1,2-CTD. Keywords Catechol 1,2-dioxygenase · Candida albicans TL3 · MALDI-TOF mass analysis
S.-C. Tsai · Y.-K. Li (&) Department of Applied Chemistry, National Chiao Tung University, 1001 Ta-Hseh Rd., Hsinchu, Taiwan e-mail: [email protected] S.-C. Tsai Department of Medical Technology, Yuanpei University, Hsinchu, Taiwan
Introduction Catechols are formed during biodegradation of a variety of aromatic compounds by aerobic microorganisms. The aromatic rings of catechols may be cleaved by intradiol dioxygenase via an ortho-cleavage pathway to form cis,cis-muconate or by extradiol dioxygenase via a meta-cleavage pathway to form 2-hydroxymuconic semialdehyde. These metabolites are degraded in the tricarboxylic acid cycle (Ngai et al. 1990; Chen and Lovell 1990; Aoki et al. 1990). Both types of catechol dioxygenase use nonheme iron as a cofactor (Nozaki 1979). Intradiol dioxygenases are classed into two structurally diVerent families: catechol 1,2-dioxygenases and protocatechuate 3,4-dioxygenases (3,4-PCDs), which are speciWc for catechol (or its derivatives) and hydroxybenzoates, respectively. In general, catechol 1,2-dioxygenases are dimeric proteins with identical or similar subunits. Catechol 1,2-dioxygenases are classiWed into two types according to their substrate speciWcities: type I enzymes are speciWc for catechols, alkylated catechols (catechol 1,2-dioxygenase, 1,2-CTD) (Nakai et al. 1990; Eck et al. 1991; Murakami et al. 1997; Briganti et al. 1997; Shen et al. 2004), and hydroxyquinols (hydroxyquinol 1,2-dioxygenase, 1,2-HQD) (Latus et al. 1995); type II enzymes are speciWc for chlorocatechols (chlorocatechol 1,2-dioxygenase, 1,2-ClCTD) (Broderick et al. 1991; Van der Meer et
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