DOI 10.1007/s00203-006-0187-4
O R I G I N A L P A P E R
Puri
Wcation 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 (1,2-CTD) 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 chromatog-raphy. 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 puri-Wed enzyme was a type I catechol 1,2-dioxygenase. This is the Wrst time that a 1,2-CTD from a eukaryote (Can-dida albicans) has been characterized. Peptide sequencing on fragments of 1,2-CTD by Edman degra-dation and MALDI-TOF/TOF mass analyses provided information of amino acid sequences for BLAST anal-ysis, the outcome of the BLAST revealed that this eukaryotic 1,2-CTD has high identity with a hypotheti-cal 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
Introduction
Catechols are formed during biodegradation of a variety of aromatic compounds by aerobic microor-ganisms. 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-dioxygen-ases (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-dioxy-genases are classiWed into two types according to their substrate speciWcities: type I enzymes are speciWc for catechols, alkylated catechols (catechol 1,2-dioxygen-ase, 1,2-CTD) (Nakai et al. 1990; Eck et al. 1991; Mura-kami 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 spe-ciWc for chlorocatechols (chlorocatechol 1,2-dioxygen-ase, 1,2-ClCTD) (Broderick et al. 1991; Van der Meer et al. 1993; Maltseva et al. 1994).
Catechol 1,2-dioxygenases play important roles in the degradation pathways of various aromatic com-pounds and are ubiquitous in microorganisms (Broder-ick et al. 1991; Latus et al. 1995; Sauret-Ignazi et a1.
1996; Briganti et al. 1997; Strachan et al. 1998; An et al.
2001; Shen et al. 2004; Ferraroni et al. 2004, 2005). 1, S.-C. Tsai · Y.-K. Li (&)
Department of Applied Chemistry,
National Chiao Tung University, 1001 Ta-Hseh Rd., Hsinchu, Taiwan
e-mail: ykl@cc.nctu.edu.tw
S.-C. Tsai
Department of Medical Technology, Yuanpei University, Hsinchu, Taiwan
2-CTDs from bacteria have been extensively character-ized in terms of their biochemical and structural prop-erties and their amino acid sequences (Kim et al. 1997,
2002, 2003;Eulberg et al. 1997; Feng et al. 1999; Pessi-one et al. 2001; Caposio et al. 2002; Kim et al.; Earhart et al. 2005). Various 1,2-CTD isozymes that diVer in
the composition of their subunits are found in microor-ganisms such as Pseudomonas arvilla C-1, Acinetobac-ter lwoYi K24, Frateuria sp. ANA-18, ArthrobacAcinetobac-ter sp. Ba-5-17, and Acinetobacter radioresistens (Aoki et al.
1984; Nakai et al. 1990; Kim et al. 1997; Murakami et al. 1998; Briganti et al. 2000). However, structural studies of 1,2-CTDs showed that the amino acid resi-dues at the active site are highly conserved, especially those responsible for iron binding (Eulberg et al. 1997; Ridder et al. 1998; Vetting et al. 2000).
Although there are many reports concerning 1,2-CTDs, they are almost all about enzymes from prokaryotes. Previously, we isolated a strain of yeast, Candida albicans, which uses phenol and/or formalde-hyde as its energy source, from soil at a petrochemical factory in Taiwan. It has high tolerance for phenol (up to 24 mM) and catalyzes phenol degradation through the ortho-cleavage pathway (Tsai et al. 2005). In the present study, we extended our previous Wndings by purifying and characterizing 1,2-CTD that may cleave the catechol ring derived from the phenol degradation.
Materials and methods Chemicals
Chemicals were purchased from Sigma (St Louis, MO) or Merck (Darmstadt, Germany). Yeast nitrogen base (YNB) without amino acids was obtained from Difco (Detroit, MI). All other chemicals used were reagent grade. BuVers used for various pH conditions were sodium acetate, pH 5.0–6.0; potassium phosphate, pH 6.5–8.5; Tris–acetate, pH 9.0–10.0.
PuriWcation of catechol 1,2-dioxygenase
Cultures were grown in eight 2-L Erlenmeyer Xasks at 30°C. Each Xask, containing 800 ml of YNB medium (0.67% w/v) and 10 mM phenol (Tsai et al. 2005), was inoculated with an initial OD600 of 0.01 of C. albicans TL3. Cells were harvested when the growth of the strain approached the stationary phase (OD600 of approximately 1.4). The harvested cells were washed twice with distilled water and resuspended in 34 ml of buVer A (50 mM Tris–HCl, pH 8.3, containing 5 mM -mercaptoethanol) and then disrupted by sonication.
After centrifugation to remove cell debris, the superna-tant was subjected to protein precipitation. The precip-itant obtained from 50 to 70% saturation of ammonium sulfate was collected and resuspended in buVer A (3 ml) for gel Wltration chromatography (Sephadex G-75, 2 £ 80 cm). The column was eluted with the same buVer at a Xow rate of 0.1 ml/min. Fractions, eluted from 610 to 680 min, with 1,2-CTD activity were pooled and concentrated by centrifugal ultraWltration (Amicon MWCO 10 kDa, Millipore). All the concen-trated sample (5 ml) was further loaded onto the HiTrap Q Sepharose column (5 ml £ 2; Amersham Bioscience, Uppsala, Sweden), pre-equilibrated with buVer A. Chromatography was performed with a linear gradient from 0 to 0.2 M of (NH4)2SO4 in buVer A at a Xow rate of 0.5 ml/min. The active fractions, eluted at 0.15–0.16 M of (NH4)2SO4, were analyzed on enzyme purity by SDS-PAGE and pooled for further study. All puriWcation steps were carried out at 4°C.
Determination of protein concentration
Protein concentration was determined using bovine serum albumin as a standard by the method of Brad-ford (BradBrad-ford 1976), and Scopes method (Scopes
1974) was also used to determine protein concentration in the analysis of iron content.
Determination of molecular weight
The molecular weight of the subunit of 1,2-CTD was determined using 12.5% SDS-PAGE as described by Laemmli (Laemmli 1970). The molecular weight of the native protein was determined using gel Wltration on a Sephadex G-75 column and a series of standard pro-teins.
A more precise estimate of subunit molecular weight was obtained using electrospray ionization mass spectrometry (ESI-MS) with a quadrupole time-of-Xight mass spectrometer (Q-TOF, Micromass, UK). The quadrupole mass analyzer scanned mass-to-charge ratios (m/z) from 100 to 2,500 units with a scan step of 2 s and an interscan of 0.1 s per step. In the ESI-MS experiment, we used the quadrupole scan mode under a capillary needle at 3 kV, a source block temperature of 80°C and a desolvation temperature of 150°C. Between 5–10g of protein in desalted form were used for MS analysis.
Enzyme activity assays
1,2-CTD activity was determined by measuring the increase of absorbance at 260 nm, corresponding to the
formation of products from all tested substrates except for hydroxyquinol and protocatechuate, for which wavelengths of 245 nm and 270 nm were employed, respectively (Varga and Neujahr 1970; Bull and Ballou
1981; Strachan et al. 1998). One unit of 1,2-CTD was deWned as the amount of enzyme needed to produce 1mol of product per minute. The standard assay of enzyme activity was performed by addition of 2l (68g/ml) of 1,2-CTD to 998 l of assay solution (buVer A containing 20 M FeSO4 and 0.1 mM of cate-chol) at 25°C. Kinetic parameters, kcat and Km, were estimated from the double reciprocal plots of speciWc rate versus substrate concentration. To investigate the thermal and pH stabilities of the enzyme and the eVects of various metal ions on 1,2-CTD activities, the enzyme was pre-incubated at various temperatures, pH values, or metal ions for 30 min. The residual enzyme activity was subsequently measured with standard assay. Iron analysis
This enzyme was nitrated in 6N HNO3 and then the iron content of the protein was determined by induc-tively coupled plasma-mass spectrometry (ICP-MS) (Agilent 7500a, USA). All glassware was acid-washed to avoid contamination with iron.
Determination of amino acid sequence
For amino sequence analysis, the puriWed 1,2-CTD was digested by trypsin (Promega Co.) in the condition of 0.2 M of ammonium bicarbonate containing 0.02% Tween-20 at 37°C for 16 h. The trypsinized peptides were separated by reverse phase HPLC using a reverse-phase column (Waters, u Bondapak C18, 3.9 £ 300 mm) with two linear gradients of acetonitrile containing 0.1% TFA (0–20% and 20–100% in 10 and 80 min, respectively). Each peak was manually col-lected and used for Edman sequencing (Procise 494 sequencer, Applied Biosystems, USA).
Trypsinized peptides were also eluted onto the MALDI target plate using 50% acetonitrile containing 0.5% TFA and 10 mg/ml -cyano-4-hydroxycinnamic acid. Sample peptides were analyzed by TOF-MS and TOF-MS/MS (4700 proteomics analyzer, Applied Bio-systems, USA). For peptide sequencing by MS/MS analysis, collision-induced dissociation was performed using air as the collision gas and the collision energy was set to 1 kV. The resulted mass spectra were ana-lyzed by using the Mascot program to search in NCBI nonreductant database and by using DeNovo Explores (TM) software (Version 3.5) to predict the amino acid sequence.
Results and discussion PuriWcation of 1,2-CTD
In our previous study, we have demonstrated that the presence of phenol in cultural medium is essential for the induction of 1,2-CTD activity. The optimal induc-tion was found when C. albicans TL3 was cultivated in a medium containing 10 mM phenol (Tsai et al. 2005). In this study, similar condition, shown in the experi-mental section, was employed to produce 1,2-CTD. PuriWcation of 1,2-CTD from cell-free extracts was per-formed using ammonium sulfate precipitation and Sephadex G-75 gel Wltration followed by HiTrap Q Sepharose column chromatography as described inMaterials and methods. Yields for each step of the puriWcation process are summarized in Table1. Forty-fold puriWcation of this 1,2-CTD was achieved with a yield of 33%. The speciWc activity of the puriWed enzyme was 63.0 units per mg protein. Gel-Wltration analysis using a Sephadex G-75 column showed that the puriWed enzyme eluted at a position corresponding to a molecular weight of about 64 kDa. After SDS-PAGE, the puriWed enzyme appeared as a single band with a molecular weight of 31 kDa (Fig.1), suggesting that the enzyme is a dimeric protein. The molecular weight of the subunit of 1,2-CTD was measured by ESI-MS to be 31,994 § 2 Da (Fig.2), which is consis-tent with the results of the SDS-PAGE analysis. The dimeric nature of this eukaryotic 1,2-CTD is similar to that of bacteria, which have molecular weights of 30,500–34,000 Da per subunit (Eck and Bettler 1991; Neidle et al. 1988).
Characterization of 1,2-CTD
The 1,2-CTD of C. albicans TL3 showed considerable activity towards catechol and 4-methylcatechol (18% of that of catechol), but no signiWcant activity towards other catechol derivatives such as 3-methycatechol, 4-chlorocatechol, 4-carboxycatechol, and hydroxyqui-nol. The products of the enzymatic reaction were ana-lyzed and conWrmed by negative mode of ESI-MS and tandem MS (data not shown). The catalytic product of 1,2-CTD towards catechol (or 4-methylcatechol) showed a signal at m/z 185 (or 199), which is consis-tent with the molecular weight of disodium muconate (or disodium 3-methylmuconate). The tandem mass analysis of m/z 185 showed the fragments with m/z 140 and 167, which were found to be completely iden-tical to the fragment pattern of pure cis, cis-disodium muconate. Similarly, the MS/MS fragments derived from the peak of m/z 199 were 154 and 181, which is
14 amu larger than those from disodium muconate. Thus, we concluded that the catalytic end product derived from 4-methylcatechol (m/z 199) is disodium
3-methylmuconate. Because of its substrate speci Wc-ity, we suggest that the 1,2-CTD of C. albicans TL3 possesses characteristics similar to those of Acineto-bacter sp. (Caposio et al. 2002; Kim et al. 2003), Pseu-domonas arvilla C-1 (Nakai et al. 1990), and Frateuria sp. ANA-18 (Aoki et al. 1984), which are classiWed as type I 1,2-CTDs. The kcat and Km values of 1,2-CTD for catechol were 28 s¡1 and 9.3M, respectively. When 4-methylcatechol was used as substrate, the kcat value (5.6 s¡1) was only 20% of that of catechol and the Km value (21.5M) was about 2.3-fold larger than that of catechol.
The optimal temperature and pH of 1,2-CTD from C. albicans TL3 was 25°C and pH 8.0, respectively. The optimal pH of this enzyme is similar to that of 1,2-CTDs that have been isolated from Pseudomonas sp. (Nakai et al. 1988; Briganti et al. 1997) and Acinet-obacter sp. (Kim et al. 2003), but lower than that iso-lated from Rhizobium leguminosarum (Chen and Lovell 1990), Rhizobium trifolii (Chen et al. 1985), and Rhodococcus rhodochrous (Strachan et al. 1998), which are optimally active at pH 9.0–9.5. The puriWed enzyme was stable (maintaining > 85% activity) for at least 30 min at the pH range of 7.0–9.0, whereas the stability of enzyme greatly decreased in the pH
Fig. 1 SDS-PAGE analysis of 1,2-CTD from C. albicans TL3 in
various steps of puriWcation. Lane M, protein markers; Lane 1, crude cell extract, 170g protein; Lane 2, 50–70% (NH4)2SO4
precipitation, 26g protein; Lane 3, after chromatography on a G-75 column, 4g protein; Lane 4, after chromatography on a Hi-Trap Q Sepharose column, 2.5g protein
M 1 2 3 4 115 kDa 91 kDa 62 kDa 46 kDa 36 kDa 26 kDa 19 kDa 198 kDa
Fig. 2 The mass spectrum of
the puriWed 1,2-CTD from C. albicans TL3 (inset) and the deconvolution of the spectrum to give a molar mass of 31,994 amu
Table 1 PuriWcation of 1,2-CTD from Candida albicans TL3
Step Fraction Volume
(ml) Proteins (mg/ml) SpeciWc activity (units/mg) PuriWcation fold Yield (%) (enzyme activity) 1 Crude extract 34 2.89 1.57 1 100 2 (NH4)2SO4 ppt, 50–70% 3 3.31 12.7 8.1 81.5
3 Eluate, Sephadex G-75 column 5 0.52 25.6 16.3 42.6
condition out of this range (Fig.3). The enzyme was found to be stable when it was kept at temperature lower than 40°C (Fig.4). The biochemical properties of 1,2-CTD from C. albicans TL3 are summarized in Table2.
The eVects of metal salts and chelating agents on the activity of this enzyme were investigated. The results are shown in Table3. The activity of this enzyme towards catechol was minimally aVected by FeSO4, FeCl3, CuSO4, CoCl2, MnSO4 or EDTA at concentra-tions of up to 0.1 mM, whereas the addition of 0.1 mM AgNO3, CuCl, HgCl2 or PbSO4 inhibited the enzy-matic reaction by 41–96%. The strong inhibition (>90%) of AgNO3 towards 1,2-CTD activity was also commonly observed in bacteria, such as that from Frateuria species ANA-18 (Aoki et al. 1984) and Aci-netobacter sp. KS-1 (Kim et al. 2003).
The metal content of 1,2-CTD was determined by inductively coupled plasma-mass spectrometry
(ICP-MS). The concentration of iron in the puriWed enzyme was estimated to be 0.0243 (§0.0012) per 0.0127 (§0.0005) nmol (determined by Bradford method) or 0.0133 (§0.0007) nmol (determined by Scopes method) of the protein. These results suggest that the 1,2-CTD of C. albicans TL3 contains one iron per sub-unit. The iron content of this 1,2-CTD is similar to that of Acinetobacter calcoaceticus (Neidle and Ornston
1986) and Rhizobium trifolii TA1 (Chen et al. 1985), but diVers from that of Brevibacterium fuscum (Nakazawa 1963), Pseudomonas sp. (Nakai et al. 1988; Briganti et al. 1997), Rhizobium leguminosarum biovar viceae USDA2370 (Chen and Lovell 1990), and Rhodococcus rhodochrous NCIM13259 (Strachan et al. 1998), which possess only one iron molecule in the dimeric form of the protein molecule.
Amino acid sequence analysis
Since the eukaryotic 1,2-CTD has not yet been exten-sively studied, N-terminal sequence by Edman degra-dation was performed. Unfortunately, the puriWed enzyme seemed to be N-terminal blocked, a feature
Fig. 3 The pH stability of 1,2-CTD from C. albicans TL3. The
en-zyme was incubated at pH 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, and 10.0 for 30 min at 25°C. The residual activity of enzyme was determined by the standard assay and compared with the activity of the enzyme kept at 25°C, pH 8.3
0 20 40 60 80 100 120 3 4 5 6 7 8 9 10 11 12 pH R e la ti ve ac ti vi ty ( % of c o n tr o l)
Fig. 4 Thermal stability of 1,2-CTD from C. albicans TL3. The
enzyme was incubated at 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70°C for 30 min at pH 8.3. The residual activity of enzyme was determined by the standard assay and compared with the activity of the enzyme kept at 25°C, pH 8.3
0 10 20 30 40 50 60 70 80 90 100 110 120 0 10 20 30 40 50 60 70 Temperature (°C) Relative activity ( % of control)
Table 2 The properties of catechol 1,2-dioxygenase from
Candida albicans TL3 Molecular weight (Da)
SDS-PAGE 31,000
ESI-MS 31,994
Gel Wltration 64,000
Iron content (mol/mol enzyme) 2
Optimal pH 8.0
Optimal temperature 25°C
Km for catechol, 4-methylcatechol 9.3M, 21.6 M
kcat for catechol, 4-methylcatechol 28 s ¡1
, 5.6 s¡1
Table 3 EVects of some metal ions and compounds on catechol
1,2-dioxygenase activity from Candida albicans TL3 for catechol
Data are expressed as mean § standard deviation (n = 3) Substance Remaining activity (%)
0.01 mM 0.1 mM None 100 100 AgNO3 7 § 2 4 § 2 CuCl 72 § 4 59 § 1 FeSO4 109 § 10 95 § 5 FeCl3 115 § 9 86 § 4 MnSO4 94 § 5 113 § 6 CuSO4 95 § 3 93 § 2 CoCl2 97 § 12 92 § 5 HgCl2 85 § 1 36 § 2 PbSO4 81 § 3 25 § 4 EDTA 99 § 7 101 § 9
that is not exhibited by most 1,2-CTDs of bacteria (Sauret-Ignazi et al. 1996; Briganti et al. 1997; Pessione et al. 2001; Kim et al. 2003). A Blast, based on the MALDI-TOF mass spectrometric analysis of the tryp-sin-digested 1,2-CTD (Fig.5), was then performed but failed to Wnd any available protein in the NCBI data-base with signiWcant similarity to 1,2-CTD. We further isolated the trypsin-digested peptides from puriWed 1,2-CTD by RP-HPLC separation. Two peptides, eluted at 15 and 44 min (designated as fragments 1 and 2, respec-tively, in Fig.6), were collected and unequivocally sequenced by an automatic Edman sequencer. The sequences of fragment 1 and 2 were highly matched to a hypothetical protein, CaO19_12036, from Candida albicans SC5314 (GenBank accession no. XM 717691) (Fig.6). In addition, MALDI-TOF/TOF mass spec-trometry (data not shown) provided high-scoring (above 80) de novo sequences of two fragments (frag-ments 3 and 4 in Fig.6) with m/z of 932 Da and 1,199 Da in Fig.5 and these sequences also appeared to be highly identical with this hypothetical protein. Therefore, we suggested this hypothetical protein CaO19_12036 of C. albicans SC5314 (XP_722784 XP_431250) should be 1,2-CTD. It is possible that may have other gene(s) in C. albicans encoding 1,2-CTD(s). However, since the complete genome of this strain has not yet available, this argument cannot be evaluated at the current stage.
Conclusion
To the best of our knowledge, this is the Wrst report on the puriWcation and characterization of 1,2-CTD from eukaryotic cell(s). The 1,2-CTD from C. albicans TL3 had substrate speciWcity similar to that of type I CTD from bacteria, and was similar to bacterial 1,2-CTDs in molecular weight, iron content, optimal pH and metal ion eVects. Based on the analyses of MALDI-TOF mass spectrometry and Edman sequenc-ing reported herein, we conWrmed that the hypotheti-cal protein CaO19_12036 from C.albicans SC5314 is indeed 1,2-CTD.
Acknowledgments This work was supported by the National
Science Council of Taiwan, the Center of Interdisciplinary Molecular Science of the National Chiao Tung University and the MOE ATU program. We also thank Ms. Chih-Yu Cheng for assistance with the mass spectrometric analysis.
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