Chapter 4 Purification and characterization of a catechol 1,2-dioxygenase from a
4.4 Results and Discussion
4.4.2 Characterization of 1,2-CTD
The 1,2-CTD of C. albicans TL3 showed considerable activity towards catechol and 4-methylcatechol (18% of catechol), but no significant activity towards other catechol derivatives such as 3-methycatechol, 4-chlorocatechol, 4-carboxycatechol,
96
and hydroxyquinol (Table 4-2). ESI tandem mass spectrometric analysis showed that the products of the reaction between 1,2-CTD and catechol or 4-methylcatechol were disodium muconate and 3-methyl-disodium muconate, respectively (Fig. 4-6).
Because of its substrate specificity, we suggest that the 1,2-CTD of C. albicans TL3 possesses characteristics similar to those of Acinetobacter sp. (Caposio et al. 2002;
Kim et al. 2003), Pseudomonas arvilla C-1 (Nakai et al. 1990), and Frateuria sp.
ANA-18 (Aoki et al. 1984), which are classified as type I 1,2-CTDs. The kcat and Km
values of 1,2-CTD for catechol were 28 s-1 and 9.3 µM, 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 (21.5 µM) was about 2.3-fold larger than that of catechol (Fig. 4-7 and Fig. 4-8). This result showed that the enzyme has higher affinity and catalytic activity for catechol than 4-methylcatechol.
The optimal temperature and pH of 1,2-CTD from C. albicans TL3 was 25 °C and pH 8.0, respectively (Fig. 4-9 and Fig. 4-10). 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 Acinetobacter sp. (Kim et al. 2003), but lower than that isolated 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 enzyme was found to be stable when
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it was kept at temperature lower than 40 °C (Fig. 4-11). The purified enzyme was stable (maintaining > 85% activity) for at least 30 min at the pH range of 7.0 to 9.0, whereas the stability of enzyme greatly decreased in the pH condition out of this range (Fig. 4-12). The biochemical properties of 1,2-CTD from C. albicans TL3 are summarized in Table 4-3.
The effects of metal salts, chelating agents, and sulfhydryl agents on the activity of this enzyme were investigated. The results are shown in Table 4-4. The activity of this enzyme for catechol was minimally affected by FeSO4, FeCl3, CuSO4, CoCl2, MnSO4 or EDTA at concentrations of up to 0.1 mM, whereas the addition of 0.1 mM AgNO3, CuCl, HgCl2 or PbSO4 inhibited the enzymatic reaction by 41%–96%.
Interestingly, the presence of β-mercaptoethanol (5 mM) enhanced activity by 254%.
The presence of Ag+ andβ-mercaptoethanol inhibited and enhanced obviously this enzyme activity, respectivity, it seems that sulfhydryl group of cysteine residue(s) play an important role in 1,2-CTD activity from C. albicans TL3, which agree well with Acinetobacter sp. KS-1 (Kim et al. 2003) and Frateuria species ANA-18 (Aoki et al. 1984).
The metal content of 1,2-CTD was determined by inductively coupled plasma-mass spectrometry (ICP-MS). The concentration of iron in the purified
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enzyme was estimated to be 0.0243 (±0.0012) nmole per 0.0127 (±0.0005) nmole (determined by Bradford method) or 0.0133 (±0.0007) nmole (determined by Scopes method) of the protein. These results suggest that the 1,2-CTD of C. albicans TL3 contains one iron per subunit. 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 differs 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.
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4.5 Conclusion
To the best of our knowledge, this is the first report on the purification and characterization of 1,2-CTD from eukaryotic cell(s). The 1,2-CTD from C. albicans TL3 had substrate specificity similar to that of type I 1,2-CTD from bacteria. From the result of metal ions and β-mercaptoethanol effect on enzyme activity, which is presumably sulfhydryl group of cysteine(s) may be involved in this enzyme activity.
In addition, this enzyme displayed a high level of homology with bacteria 1,2-CTDs in molecular mass, iron content, temperature, pH and metal ion sensibilities.
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4.6 Referances
Aoki K, Konohana T, Shinke R (1984) Two catechol 1,2-dioxygenase from aniline-assimilating bacterium, Frateuria species ANA-18. Agric Biol Chem 48 (8):2097-104.
Aoki K, Nakanishi Y, Murakami S, Shinke R (1990) Microbial metabolism of aniline through a meta-cleavage pathway: isolation of strains and production of catechol 2,3-dioxygenase. Agric Biol Chem 54:205-6.
Bradford MM (1976) A rapid and sensitive methods for the quantitation of microgram quantities of protein utilizing the principle for protein-dye binding. Anal Biochem 72:248-54.
Briganti F, Pessione E, Giunta C, Scozzafava A (1997) Purification, biochemical properties and substrate specificity of a catechol 1,2-dioxygenase from a phenol degrading Acinetobacter radioresistens. FEBS Lett 416:61-4.
Broderick JB, O,Halloran TV (1991) Overproduction, Purification, and characterization of chlorocatechol dioxygenase, a non-heme iron dioxygenase with broad substrate tolerance. Biochemistry 30:7349-57.
Bull C, Ballou DP (1981) Purification and properties of protocatechuate 3,4-dioxygenase from Pseudomonas putida. J Biol Chem256: 12673-80.
Caposio P, Pessione E, Giuffrida G, Conti A, Landolfo S, Giunta C, Gribaudo G (2002) Cloning and characterization of two catechol 1,2-dioxygenase genes fromAcinetobacter radioresistens S13. Res Microbiol 153:69-74.
101
Chen YP, Glenn AR, Dilworth MJ (1985) Aromatic metabolism in Rhizobium trifolii
⎯ catechol 1,2-dioxygenase. Arch Microbiol 141:225-8.
Chen YP, Lovell CR (1990) Purification and properties of catechol 1,2-dioxygenase from Rhizobium leguminosarum biovar viceae USDA2370. Appl Environ Microbiol 56:1971-3.
Eck R, Bettler J (1991) Cloning and characterization of a gene coding for the catechol
1,2-dioxygenase of Acinetobacter sp. mA3. Gene 123:87-92.
Kim SI, Song SY, Kim KW, Ho EM, Oh KH (2003) Proteomic analysis of the benzoate degradation pathway in Acinetobacter sp. KS-1. Res Microbiol;154:697-703.
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 1970 (London) 227:680-5.
Maltseva OV, Solyanikova IP, Golovleva LA (1994) Chlorocatechol 1,2-dioxygenasefrom Rhodococcus erythropolis 1CP. Kinetic and immunochemical comparison with analogous enzymes from gram-negative strains.Eur J Biochem 226:1053-61.
Murakami S, Kodama N, Shinke R, Aoki K (1997) Classification of catechol 1,2-dioxygenase family: sequence analysis of a gene for the catechol 1,2-dioxygenase showing high specificity for methylcatechols from Gram+ aniline-assimilating Rhodococcus erythropolis AN-13. Gene 185:49-54.
Nakai C, Nakazawa T, Nozaki M (1988) Purification and properties of catechol
102
1,2-dioxygenase (pyrocatechase) from Pseudomonas putida mt-2 in comparison with that from Pseudomonas arvilla C-1. Arch Biochem Biophys 267:701-13.
Nakai C, Horiike K, Kuramitsu S, Kagamiyama H, Nozaki M (1990) Three isoenzymes of catechol 1,2-dioxygenase (pyrocatechase), αα, αβ, and ββ, from Pseudomonas arvilla C-1. J Biol Chem 265:660-5.
Nakazawa H, Inoue H, Takeda Y (1963) Characteristics of catechol oxygenase from Brevibacterium fuscum, J Bacteriol 54:65-74.
Neidle EL, Ornston LN (1986) Cloning and expression of Acinetobacter calcoaceticus catechol 1,2-dioxygenase I structural gene catA in Escherichia coil. J Bacteriol 168:815-20.
Neidle EL, Hartnett C, Bonitz S, Ornston LN (1988) DNA sequence of the Acinetobacter calcoaceticus catechol 1,2-dioxygenase I structural gene catA:
evidence for evolutionary divergence of intradiol dioxygenase by acquisition of DNA sequence repetitions. J Bacteriol 170:4874-80.
Ridder L, Briganti F, Boersma MG, Boeren S, Vis EH, Scozzafava A, Verger C, Rietjens IM (1998) Quantitative structure/activity relationship for the rate of conversion of C4-substituted catechols by catechol-1,2-dioxygenase from Pseudomonas putida (arvilla) C1. Eur J Biochem 257:92-100.
Scopes RK (1974) Measurement of protein by spectrophotometry at 205 nm. Anal Biochem 59:277-82.
Shen XH, Liu ZP, Liu SJ (2004) Functional identification of the gene locus (ncg12319) and characterization of catechol 1,2-dioxygenase in Corybebacterium
103
glutamicum. Biotechnol Lett 26:575-80.
Strachan PD, Freer AA, Fewson CA (1998) Purification and characterization of catechol 1,2-dioxygenase from Rhodococcus rhodochrous NCIM13259 and cloning and sequencing of its catA gene. Biochem J 333:741-7.
Tsai SC, Tsai LD, Li YK (2005) An isolated Candida albicans TL3 capable of degradingphenol at large concentration. Biosci Biotechnol Biochem 69: 2358-67.
Van der Meer JR, Eggen RIL, Zehnder AJB, De Vos WM (1993) Sequence analysis of the Pseudomonas sp. Strain P51 tcb gene cluster, which encodes metabolism of chlorinated catechols: evidence for specialization of catechol 1,2-dioxygenase for chlorinated substrates. J Bacteriol 173:2425-34.
Varga JM, Neujahr HY (1970) Purification and properties of catechol 1,2-dioxygenase from Trichosporon cutaneum. Eur J Biochem 12: 427-34.
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Table 4-1. Purification of catechol-1,2-dioxygenase from C. albicans TL3.
Step Fraction Volume 3 Eluate, Sephadex G-75
column
5 0.52 25.6 16.3 42.6
4 Eluate, HiTrap Q Sepharose column
1.2 0.68 63.0 40.1 33.1
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Table 4-2. Substrate specificity of 1,2-CTD from C. albicans TL3
Substrate Relative enzyme activity (%)
Catechol 100
3-Methylcatechol 0
4-Methylcatechol 18
4-Chlorocatechol 0
4-Carboxycatechol 0
Hydroxyquinol 0
Protocatechuate 0
2,3-dihydroxybenzoic acid 0 3,4-dihydroxybenzaldehyde 0 3,4-dihydroxybenzylamine 0
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Table 4-3. The properties of 1,2-CTD from C. albicans TL3.
SDS-PAGE 31,000
ESI-MS 31,994
Molecular weight (Da)
Gel filtration 64,000
Iron content (mol /mol enzyme) 2
Optimum pH 8.0
Optimum temperature 25 °C
Stability of pH 7.0∼9.0
Stability of temperature below 40 °C
Km for catechol, 4-methylcatechol 9.3 µM, 21.6 µM kcat for catechol, 4-methylcatechol 28 s-1, 5.6 s-1
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Table 4-4. Effects of some metal ions and compounds on the activity of 1,2-CTD from C. albicans TL3 for catechol.
* The activity of the enzyme was increased to 192% and 254% of that of the
intact enzymeby the addition of 1 mM and 5 mM β-mercaptoethanol, respectively.
Data are expressed as the mean ± standard deviation (n = 3).
Remaining activity (%)
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Figure 4-1. Separation of catechol 1,2-dioxygenase from 50-70% (NH4)2SO4 ppt on a G-75column (2x80 cm). Catechol 1,2-dioxygenase (●), absorbance at 280nm (○).
0 10 20 30 40 50 60 70 80
0 4 8 12 16 20
0 1 2 3 4 5
enzyme activity (U/ml) OD280
volum e (m l)
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Figure 4-2. Separation of catechol 1,2-dioxygenase from the catechol
1,2-dioxygenase-containing fractions of G-75 column on a Q-sephadex column. Catechol 1,2-dioxygenase (●), absorbance at 280nm (○).
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Figure 4-3. Native molecular mass determination of 1,2-CTD from C. albicans TL3 by G-75 column Chromatography. A, ribonuclease A (13.7 KDa); B, chymotrypsin (25 KDa); C, ovalbumin (43 KDa); D, albumin (67 KDa); E, 1,2-CTD (64 KDa). V and V′ are elution volume for sample protein and blue dextran (2000 KDa),
respectively.
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Figure 4-4. SDS-PAGE analysis of 1,2-CTD from C. albicans TL3 in various steps of purification. Lane M, protein markers; Lane 1, crude cell extract, 170 µg protein; Lane 2, 50%–70% (NH4)2SO4 precipitation, 26 µg protein; Lane 3, after chromatography on a G-75 column, 4 µg protein; Lane 4, after chromatography on a HiTrap Q Sepharose column, 2.5 µg protein.
M 1 2 3 4 115 kDa
91 kDa 62 kDa 46 kDa
36 kDa
26 kDa
19 kDa 198 kDa
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Figure 4-5. The mass spectrum of the purified 1,2-CTD from C. albicans TL3 (inset) and the deconvolution of the spectrum to give a molar mass of 31,994 atomic mass units.
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Figure 4-6. ESI-MS/MS analysis of the product of catechols catalyzed by 1,2-CTD from C. albicans TL3. The products of the 1,2-CTD-catalyzed reaction for catechol (a) and 4-methylcatechol (b) was disodium
muconate and 3-methyl-disodium muconate, respectively.
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Figure 4-7. Kinetic property of 1,2-CTD from C. albicans TL3 for catechol.
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Figure 4-8. Kinetic property of 1,2-CTD from C. albicans TL3 for 4-methylcatechol.
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Figure 4-9. Optimal temperature of catechol 1,2-dioxygenase from C. albicans TL3.
The relative enzyme activity was determined at designated temperature in the pH 8.3 buffer.
0 20 40 60 80 100 120
0 10 20 30 40 50 60
Temperature ( ℃ )
Relative activity ( % of control)
117 0
20 40 60 80 100 120
4 5 6 7 8 9 10 11 12
pH
Relative activity (% of control)
Figure 4-10. Optimal pH of catechol 1,2-dioxygenase from C. albicans TL3. The relative enzyme activity was determined at 25 ℃ in the designated pH of buffer.
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Relative activity ( % of control)
Figure 4-11. Thermal stability of catechol 1,2-dioxygenase from C. albicans TL3.
The enzyme was incubated at indicated temperatures for 30 min at pH8.3. The remaining activity was determined by the standard assay at 25℃.
119 0
20 40 60 80 100 120
3 4 5 6 7 8 9 10 11 12
pH
Relative activity (% of control)
Figure 4-12. pH stability of catechol 1,2-duoxygenaase from C. albicans TL3. The enzyme was incubated at indicated pH for 30 min at 25℃. The remaining enzyme activity was determined by the standard assay.
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Chapter 5
Proteomic analysis of a catechol 1,2-dioxygenase from a phenol degrading Candida albicans TL3
5.1 Abstract
2-D gel analysis of purified 1,2-CTD from Candida albicans TL3 revealed five spots with similar molecular weights (~32000 Da) but different pIs. These spots were subjected to further analysis using MALDI-TOF mass spectrometry. The results of MALDI-TOF analysis suggested that all of these spots were derived from the same 1,2-CTD, presumably resulting from different degrees of post-translational modification. Peptide sequencing of the fragments of 1,2-CTD by Edman
degradation and MALDI-TOF/TOF analyses provided information on amino acid sequences for BLAST analysis. The BLAST analysis revealed that this eukaryotic 1,2-CTD is highly homologous (greater than 95%) to a hypothetical protein,
CaO19_12036, which occurs in the same Candida strain and is similar to bacterial hydroxyquinol 1,2-dioxygenase.
Keywords: 1,2-CTD, Candida albicans TL3, 2-D gel, MALDI-TOF/TOF
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5.2 Introduction
Catechols 1,2-dioxygenases play a fundamental important role in the metabolic conversion of aromatic compounds to aliphatic compounds and are ubiquitous in microorganisms (Broderick 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; Ferraroni et al. 2005). Most catechols 1,2-dioxygenases are nonheme iron enzymes and dimer form. In generally, each subunit of catechol 1,2-dioxygenase (1,2-CTD) and hydroxyquinol 1,2-dioxygenase (1,2-HQD) is composed of the 280∼310 amino acid residues, while the chlorocatechol 1,2-dioxygenase (1,2-ClCTD) is 240∼260 amino acid residues. Except from the same genera microorganisms, the homology of amino acid sequence of 1,2-CTDs, 1,2-HQDs and 1,2-ClCTDs are low.
However, structural studies of three family enzymes showed that the amino acid residues 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). Heterogeneous subunits often make 1,2-CTDs having two or three isozymes that are found in some microorganisms such as Pseudomonas arvilla C-1, Acinetobacter lwoffii K24, Frateuria sp. ANA-18, Arthrobacter 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).
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Recently, proteomic analysis of the phenolic compounds degradation pathway and related enzymes (especially to 1,2-CTDs) in bacteria is one of popular model (Giuffrida et al. 2001; Kim SI et al. 2002, 2003). Therefore, we want to use proteomic analysis to advance the realization of 1,2-CTD, which we have purified previously from C. albicans TL3.
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5.3 Experimental
5.3.1 2-D gel electrophoresis
The 1,2-CTD from C. albicans TL3 for 2-D gel electrophoresis was prepared by denaturing the purified enzyme (~30 µg) with 1 ml acetone containing 10% TCA.
The precipitated protein was collected by centrifugation. The pellet was washed several times with acetone and resuspended in 300 µl sample buffer (6 M urea, 2 M thiourea, 0.5% TritonX-100, 1% DTT, and 0.5% IPG buffer). The resulting solution was applied to an immobilized pH 3–10 and pH 4–7 nonlinear gradient strip for isoelectric focusing using IPGphor (Pharmacia). The electrophoresis was carried out at five power settings: 30 V for 12 h, 100 V for 2 h, 500 V for 2 h, 1000 V for 2 h, and 8000 V for 8 h. The second dimension was performed using 12.5% SDS-PAGE (Hoefer SE 600, Amersham Biosciences, USA). The gel was stained with Coomassie Brilliant Blue R250 (Sigma-Aldrich).
5.3.2 In-gel digestion
The stained protein spots excised from the 2-D PAGE gel were dehydrated three times using 50% acetonitrile and 0.2 M ammonium bicarbonate and then digested with trypsin (Promega) in 0.2 M ammonium bicarbonate containing 0.02%
Tween-20 at 37 °C for 16 h.
5.3.3 N-terminal protein sequencing
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The five major protein spots separated on the 2-D gel were transferred onto a PVDF membrane (Problott, Applied Biosystems, USA) at 400 mA for 1 h using a TE2 transphor electrophoresis unit (Hoefer, Amersham Biosciences, USA) with 10 mM CHAPS buffer (pH 11) containing 10% methanol. The membrane was stained with Coomassie Brilliant Blue R250 solution and washed with 50% methanol. Stained protein spots were excised from the membrane and installed in the blot cartridge of a Procise 494 sequencer (Applied Biosystems, USA) for amino acid sequencing.
5.3.4 Peptide sequencing by MALDI-TOF
Trypsinized peptides were dissolved in 0.5% TFA solution and then eluted onto the MALDI target plate using 50% acetonitrile containing 0.5% TFA and 10 mg/ml α-cyano-4-hydroxycinnamic acid. Sample peptides were analyzed by TOF-MS and TOF-MS/MS (4700 proteomics analyzer, Applied Biosystems, Framingham); UV light (355 nm) was provided by an Nd:YAG laser with a 200 Hz repetition rate. For peptide sequencing by MS/MS analysis, collision-induced dissociation was performed using air as the collision gas. The collision energy was set to 1 kV and mass spectra were analyzed using the Mascot program and the NCBI nonreductant database.
MS/MS spectra were analyzed using DeNovo Explores (TM) software (Version 3.5).
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5.4 Results and Discussion
5.4.1 MALDI-TOF analysis of the isotypes of 1,2-CTD
A 2-D gel electrophoresis was employed for analyzing the purity of 1,2-CTD.
Five major protein spots with nearly identical molecular weights (~32,000 Da) but slightly different pI values (5.3−5.7) were observed in the acidic region of the 2-D gel (Fig. 5-1, 5-2). The pI value of this enzyme was slightly higher compared to
previous pI values (4.2−5.2) of 1,2-CTDs from prokaryotic cell (Aoki et al. 1984;
Sauret-Ignazi et a1. 1996; Briganti et al. 1997; Giuffrida et al. 2001; Kim et al. 2003).
These protein spots were digested in-gel with trypsin before MALDI-TOF analyses were performed. The peptide fragmentation patterns derived from these protein spots were almost identical (Fig. 5-3), indicating that the five protein spots (isotypes) were probably derived from the same gene. The reason for the slight differences in pI between isotypes of 1,2-CTD is unknown. Although it is likely that the isotypes of 1,2-CTD resulted from post-translational modifications, further study is required to elucidate this enigma. Similarly, isotypes of 1,2-CTD were found in Acinetobacter lowffii K24 (Kim et al. 2002).
5.4.2 Amino acid sequence analysis of 1,2-CTD
To identify the amino acid sequence of 1,2-CTD from C. albicans TL3, the five
126
isotypes that separated on the 2-D gel were electroblotted onto a PVDF membrane and then subjected to N-terminal sequencing by automatic Edman degradation.
Unfortunately, all five isotypes seemed to be N-terminal blocked, a feature 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). Recently, a database of 1,2-CTDs from many microorganisms, including Acinetobacter, Bradyrhizobium, Corynebacterium, Pseudomonas, Rhodococcus, Streptomyces, has been established. However, the
Blast search of MALDI-TOF mass spectrometry showed that 1,2-CTD from C.
albicans TL3 does not have any significant similarity to proteins currently available in
the NCBI database. Therefore, we suggest that the primary structure of 1,2-CTD from C. albicans TL3 may be very different from those of bacteria.
The trypsin-digested peptides from purified 1,2-CTD were further isolated by RP-HPLC separation (Fig. 5-4) and sequenced by an automatic Edman sequencer.
Two peptides (fragments 1 and 2 in Fig. 5-7) were unequivocally sequenced.
Based on these sequences, a Blast analysis was performed. A hypothetical protein
⎯ CaO19_12036 from Candida albicans SC5314 (GenBank accession no. XM 717691) was hit with a very low E value (1e-09). In addition, MALDI-TOF/TOF mass spectrometry provided high-scoring (above 80) de novo sequences of two
fragments with m/z of 932 Da (fragment 4) and 1199 Da (fragment 3) (Fig. 5-5, 5-6).
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These sequences were identical to that of the hypothetical protein, which is
suggested to be similar to the bacterial hydroxyquinol 1,2-dioxygenase. Thus, we believe that this hypothetical protein of C. albicans SC5314 is indeed a 1,2-CTD.
Recently, Earhart et al. have determined the residues form the active sites of 1,2-CTD, 1,2-ClCTD and 1,2-HQD family from prokaryotic cell (Ferraroni et al.
2004; Ferraroni et al. 2005; Earhart et al. 2005). A multiple sequence alignment of various 1,2-CTDs revealed 1,2-CTD from C. albicans SC5314 had high conserved residues in the active site, especially in iron-binding site residues are absolutely conservation (Fig. 5-8). This result perhaps will be helpful to explain why
1,2-CTDs from C. albicans TL3. and bacteria are significantly different in sequence but with the same catalytic function. Interestingly, 1,2-CTD of C. albicans was more close to 1,2-ClCTD of R. opacus 1CP (only one residue difference : Val 254/Cys 224) than other bacterial 1,2-CTDs in residues within the active site. It is likely that this position residue (Val 254/Cys 224) maybe responsible for the
substrate selectivity difference between1,2-CTD and 1,2-ClCTD.
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5.5 Conclusion
On the basis of MALDI-TOF mass spectrometry analysis, we suggest that the five 1,2-CTD isotypes from C. albicans TL3 that were separated on 2-D gel represent various levels of post-translational modification of the same enzyme. We confirmed that hypothetical protein CaO19_12036 from C. albicans SC5314 is indeed 1,2-CTD, which is very different, in terms of primary structure, from those of bacterial enzymes.
As compared with the 1,2-ClCTD from R. opacus 1CP, the Val 254 of 1,2-CTD from C. albicans may be critical for substrate recognition. This study provides useful
genetic information, which can facilitate the gene cloning of 1,2-CTD from Candida albicans TL3.
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5.6 References
Aoki K, Konohana T, Shinke R (1984) Two catechol 1,2-dioxygenase from an aniline-assimilating bacterium, Frateuria species ANA-18. Agric Biol Chem 48 (8):2097-104.
Aoki K, Nakanishi Y, Murakami S, Shinke R (1990) Microbial metabolism of aniline through a meta-cleavage pathway: isolation of strains and production of catechol 2,3-dioxygenase. Agric Biol Chem 54:205-6.
Briganti F, Pessione E, Giunta C, Scozzafava A (1997) Purification, biochemicalproperties and substrate specificity of a catechol 1,2-dioxygenase from a phenol degrading Acinetobacter radioresisten. FEBS Lett 416:61-4.
Briganti F, Pessione E, Giunta C, Mazzoli R, Scozzafava A (2000) Purification and catalytic properties of two catechol 1,2-dioxygenase isozymes from
benzoate-grown cells of Acinetobacter radioresistens. J Protein Chem 19:709-16.
Broderick JB, O,Halloran TV (1991) Overproduction, Purification, and characterization of chlorocatechol dioxygenase, a non-heme iron dioxygenase with broad substrate tolerance. Biochemistry 30:7349-57.
Eulberg D, Golovleva LA, SchlOmann M (1997) Characterization of catechol catabolic genes from Rhodococcus erythropolis ICP. J Bacteriol 179: 370-81.
Ferraroni M, Solyanikova IP, Kolomytseva MP, Scozzafava A, Briganti F (2004) Crystal structure of 4-chlorocatechol 1,2-dioxygenase from the
130
chlorophenol-utilizing gram-positive Rhodococcus opacus 1CP. J Biol Chem 279:27646-55.
Ferraroni M, Seifert J, Travkin VM, Thiel M, Kaschabek S, Scozzafava A, Golovleva L, Schlomann M, Briganti F (2005) Crystal structure of the hydroxyquinol 1,2-dioxygenase from Nocardioides simplex 3E, a key enzyme involved in polychlorinated aromatics biodegradation. J Biol Chem 280:21144-54.
Giuffrida MG, Pessione E, Mazzoli R, Dellavalle G, Braello C, Conti A, Giunta C (2001) Media containing aromatic compounds induce peculiar proteins in Acinetobacter radioresistens, as revealed by proteome analysis.
Electrophoresis 22:1705-11.
Kim SI, Leem SH, Choi JS, Chung YH, Kim S, Park YM, Lee YN, Ha KS (1997)
Cloning and characterization of two catA genes in Acinetobacter lwoffii K24. J
Cloning and characterization of two catA genes in Acinetobacter lwoffii K24. J