Expression, purification, and sequence analysis of catalase-1 from the soil bacterium Comamonas terrigena N3H

Protein Expression and PuriWcation 36 (2004) 115–123 www.elsevier.com/locate/yprep

Expression, puriWcation, and sequence analysis of catalase-1 from the soil bacterium Comamonas terrigena N3H Marcel Zámocký,a,b,¤ Jana Godobíková,a Juraj Ganperík,a Franz Koller,c and Bystrík Poleka a

Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, SK-84551, Bratislava, Slovakia b Institute of Food Technology, University of Agricultural Sciences, Muthgasse 18, A-1190, Vienna, Austria c Institute of Biochemistry and Molecular Cell Biology, University of Vienna, Dr. Bohrgasse 9, A-1030 Vienna, Austria Received 4 February 2004, and in revised form 2 March 2004 Available online 20 April 2004

Abstract Catalases are essential components of the cellular equipment to cope with oxidative stress. We have puriWed and characterize herein the most abundant heme-containing catalase-1 from the soil bacterium Comamonas terrigena N3H. This oxidative stressinduced enzyme was isolated from exponential phase cells grown in the presence of peroxyacetic acid. We have used consecutive steps of hydrophobic, molecular sieve, and ion exchange chromatography to achieve a high state of purity for this metalloenzyme. The puriWed sample of catalase exhibited a speciWc catalatic activity of 55,900 U/mg, allosteric behavior in peroxidic reaction, a broad pH optimum, and a rather atypical electronic spectrum. The sample of highest purity was subjected to mass spectrometry analysis. The molecular weight of the subunit of this homodimeric protein was determined as 55,417 Da. The Qq-TOF mass analysis method allowed us to sequence short tryptic fragments of this catalase. Five such fragments with a total length of 57 amino acids together with several enzymatic properties allowed the classiWcation of this hydroperoxidase as belonging to clade III of monofunctional catalases. The highest sequence similarity is with the catalase from Vibrio Wscheri. The presented results imply the signiWcance of this inducible enzyme in the prevention of toxic eVects of oxidative stress for bacterial cells.  2004 Elsevier Inc. All rights reserved.

Reactive oxygen species (ROS) formed as by-products in the metabolism represent a serious problem for all organisms utilizing molecular oxygen as the Wnal electron acceptor. ROS can damage all important components of the cell including DNA and proteins, and several defence mechanisms were evolved to alleviate the harmful eVects of the oxidative stress [1]. Among several potentially harmful ROS, hydrogen peroxide is probably the most abundant one, regularly occurring in the cells and also in their environment. Hydrogen peroxide is formed intracellularly as by-product in reactions catalyzed by numerous oxidases, mainly those belonging to Xavoenzymes [2]. Catalases (E.C. 1.11.1.6) are enzymes evolved to decompose hydrogen peroxide eYciently and rapidly to oxygen and water [3]. They are spread among all aerobically living organisms and they are also present in some facultative anaerobes [4]. The reaction mecha¤

Corresponding author. Fax: +00-4212-5930-7416. E-mail address: [email protected] (M. Zámocký).

1046-5928/$ - see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2004.03.001

nism of these oxidoreductases was studied in detail [5]. It is based on the heterolytic cleavage of the peroxidic bond present in hydrogen peroxide and also some organic peroxides. A highly reactive reaction intermediate termed compound I is formed from ferricatalase in a rapid reaction with the peroxide. In addition to hydrogen peroxide, also various 1- and 2-electron donors can be used for the reduction of Compound I back to ferricatalase thus representing the peroxidatic mode of catalase reaction [5]. In prokaryotes, catalases are very abundant enzymes and some bacteria express several isoenzyme forms [6]. We have investigated the response of Comamonas terrigena N3H to various forms of oxidative stress [7] and have isolated the most abundant inducible form of catalase in this soil microorganism. From the puriWed catalase-1, the sequence of the two conserved regions located in the distal and proximal side of the prosthetic heme group was determined. According to these partial sequences, and supported by the observed biochemical and physical properties, we were

116

M. Zámocký et al. / Protein Expression and PuriWcation 36 (2004) 115–123

able to classify catalase-1 from C. terrigena as member of the group of typical monofunctional catalases. Obtained properties can help us to understand the behavior of related bacterial catalases, some of them from pathogenic microorganisms.

Materials and methods Bacterial strain The bacterium C. terrigena N3H was obtained from the bacterial collection of the Institute of Molecular Biology, Slovak Academy of Sciences. Originally, it was isolated from the contaminated soil of Middle Slovakia [8]. Between the experiments it was maintained on agar plates with minimal medium at 28 °C. Expression of catalase-1 The bacterial culture was cultivated in a liquid medium, containing 1% meat extract, 1% peptone, and 0.5% NaCl, pH 7.2. The expression of catalase was induced by the addition of peroxyacetic acid (PAA) to the Wnal concentration of 0.5 mM in the middle exponential phase of growth at 28 °C as monitored by determining the absorbance of growing culture at 610 nm. We have shown before that the bacterial culture under given conditions produces the highest yield of catalase-1 with only very low level of other catalase isozymes [7]. The cells expressing the inducible catalase-1 were harvested after 1 h of incubation at 28 °C by centrifugation for 10 min. at 5000g. Method of isolation of a typical intracellular catalase The cells (approximately 26 g of wet weight) were homogenized in 450 ml of the buVer containing 20 mM Tris–HCl, pH 7.5, with glass beads of 0.2 mm diameter. Catalase was separated from other intracellular proteins with fractionated ammonium sulfate precipitation. Most of the catalase activity was observed in the fraction obtained at 0% ! 45% (w/v) saturation. This pellet was resuspended in a minimal volume of 20 mM Tris–HCl buVer, pH 7.5, containing 0.85 M ammonium sulfate. The supernatant after centrifugation was applied to a column of Spheron 100.000 (Lachema, Czechoslovakia). Spheron is a hydrophobic matrix of [poly(hydroxyethylmethacrylate)] and we used a column of 2.6 £ 6 cm equilibrated with 20 mM Tris–HCl, pH 7.5, containing 1.7 M ammonium sulfate. Elution of the bound proteins from this column was achieved by applying a linear gradient of 1.7 M ! 0.0 M ammonium sulfate in 20 mM Tris– HCl, pH 7.5. The fractions showing the highest enzyme activity were pooled and subjected to a further chromatography step on a Superose 12 prep grade column

(Pharmacia, “Superose P12,” 2.5 £ 40 cm) equilibrated with the above mentioned buVer. The active fractions were applied on a column of Q Sepharose Fast Xow (Pharmacia) with a bed volume of 10 cm3 in 20 mM Tris–HCl, pH 7.5, and eluted with a 100 ml gradient of 0 ! 0.4 M NaCl in the same buVer. Pooled active fractions were diluted 4-fold in 20 mM Tris–HCl, pH 7.5, and re-chromatographed on a column of Mono Q HR 5/ 5 (Pharmacia) with a 40 ml gradient of 0.0 ! 0.4 M NaCl in 20 mM Tris–HCl, pH 7.5. The major portion of catalase was eluted in approximately 70% of this gradient. Polyacrylamide gel electrophoresis and staining of the gels To reveal the level of protein expression and puriWcation both, native gradient and SDS gel electrophoresis were performed. SDS–polyacrylamide gel electrophoresis (8% in acrylamide) was run as described by Laemmli [9]. Linear gradients of acrylamide (4–18%) were applied in native polyacrylamide gel electrophoresis. Protein staining was performed with 0.25% (w/v) Coomassie brilliant blue R-250 in 30% methanol and 10% acetic acid. Catalase staining was performed according to Woodbury [10], based on the formation of Berlin-Blue. Protein bands showing catalase activity appeared as white spots in a dark green background of the gel. Enzymatic assays Catalatic activity was determined at room temperature by monitoring the decomposition of H2O2 at 240 nm [11]. Peroxidatic activity with 2-electron donors was determined at 340 nm in an assay described before with a continuous production of hydrogen peroxide [12]. To determine the peroxidatic activity with 1-electron donors, a similar H2O2 producing system was applied. Oxidation of the respective donor 2,2⬘-azino-bis-(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) or guaiacol was observed at 660 or 436 nm, respectively. Spectrophotometric analysis The electronic spectrum of isolated catalase was recorded at room temperature between 800 and 200 nm with a scan speed of 60 nm/min to observe the typical patterns of the heme prosthetic group in the protein environment. Mass spectrometry analysis Using the peptide mass Wngerprinting technique an array of peptide masses from the enzymatic digest of the protein isolated from a native gradient gel was recorded. These masses were then compared to theoretical mass values in the databases. The matrix assisted laser desorption/ionization technique generated almost only singly

M. Zámocký et al. / Protein Expression and PuriWcation 36 (2004) 115–123

charged ion species and hence this was the preferred ionisation technique for the peptide mass Wngerprinting experiments with Catalase-1. As a second step of the MALDI investigation structural information about the obtained peptides was gained in a postsource decay. PE Biosystems Qq-TOF device was used to carry out the catalase peptide sequencing experiments. In the nanospray technique, sample volumes of 1–2
l, with a Xow rate of 50 nl/min, were used. During the electrospray ionisation, multiply charged ions were formed and they were fragmented in a collision cell. The created fragment spectrum—in contrast to the MALDI PSD spectra—revealed sequence fragments of catalase-1 from C. terrigena. Sequence analysis Sequence alignment of the peptide fragments obtained by mass spectrometry analysis with similar sequence motifs from GenBank database was performed with ClustalX version 1.81 [13]. The parameters of the alignment were: gap opening penalty 10.0; gap extension penalty, 0.2; and Gonnet point-accepted mutation (PAM) 250 series protein-weight matrix. Search for similar motifs was performed also with Blast (basic local alignment search tool, [14]) against all available sequence databases with parameters set according to recommendations of the authors.

117

Fig. 1. Behavior of a sample of puriWed (see Materials and methods) catalase-1 from Comamonas terrigena N3H in a native gradient PAGE. (A) Stained with Coomassie blue; (B) stained for catalase activity (St HMW reXects bovine liver catalase present in a commercial molecular weight standard mixture showing a).

exchange chromatography Wnally led to a sample with a 193-fold enrichment and a speciWc activity of 55,900 U/ mg as is obvious from the puriWcation Table 1. During the puriWcation procedure in each step we collected, as a rule, only those fractions that had signiWcantly higher speciWc activity as compared with the previous step. Therefore, e.g., a lowered yield occurred in the step of hydrophobic chromatography on Spheron. The Wnal puriWed sample showed only one single band corresponding to the size of a catalase subunit (56 kDa) on a SDS–polyacrylamide gel (see Fig. 1A). This sample was also applied on a native gradient gel followed by speciWc activity staining under non-denaturing conditions (Fig. 1B). The observed band indicates the presence of a catalytically active dimeric form.

Results and discussion Properties of puriWed catalase-1 Isolation of the typical intracellular catalase-1 In our previous investigations, we have detected the presence of three diVerent catalase isoenzymes in C. terrigena that are diVerentially regulated [7]. In the present work, we focus on the characterization of the major inducible form named catalase-1. This isoenzyme is present also in non-induced cells, but after addition of 0.5 mM peroxyacetic acid to the cultivation medium it is the most abundant form. The reWned method of isolation of this typical intracellular catalase based on combination of hydrophobic, molecular sieve, and ion

The rather high value for the speciWc catalatic activity of 55,900 U/mg falls in the range that is typical for monofunctional heme-containing catalases. When compared with the performance of catalases from mammals or yeast, this enzyme has no signiWcant activity with 1electron donors and only rather low with 2-electron donors (on average 33 mU/mg with ethanol and 5.2 mU/ mg with 1-propanol) under comparable conditions. The calculated ratio of catalatic/peroxidatic activity of 14 £ 106 for ethanol is about three orders of magnitude larger than the corresponding number obtained for

Table 1 PuriWcation of catalase-1 from Comamonas terrigena N3H Fraction

Volume (ml)

Protein (mg/ml)

Crude extract 45% (NH4)2SO4 supernatant Spheron 100.000 Superose P 12 Q Sepharose fast Xow Mono Q HR5/5

465.0 31.0

2.40 6.10

15.8 16.0 11.0 0.5

3.35 0.80 0.40 0.25

SpeciWc activity (U/mg)

Total units (U)

Yield (%)

290 1300

319,900 237,800

100 74

2100 7200 14,600 55,900

112,700 92,500 64,200 7000

This puriWcation was performed with 26.02 g harvested cell suspension.

35 29 20 2.2

PuriWcation (-fold) 1.0 4.5 7.3 24.9 50.3 192.8

118

M. Zámocký et al. / Protein Expression and PuriWcation 36 (2004) 115–123

other eukaryotic peroxisomal catalases [3] and supports the classiWcation of this enzyme as a monofunctional catalase, hydrogen peroxide being the preferred substrate in vivo. Interestingly, the behavior of catalase-1 with short aliphatic alcohols as 2-electron donors can be explained as exhibiting allosteric eVects (data not shown). In Table 2, the corresponding Hill coeYcients and K0.5 constants are presented. Substrate aYnities are in the same range as reported for related enzymes, and the apparent Hill coeYcients fairly agree with the observed dimeric nature of the active enzyme. Still, positive cooperativity between active sites of multimeric catalases is very uncommon, though there are observations that indicate mutual interaction between sites [15]. Table 2 Reactivity of catalase-1 from Comamonas terrigena with 2-electron donorsa Substrate

Ratio Ukat/ Upox

app. Hill coeYcient

app. K1/2 (M)

Ethanol 1-Propanol

4,530,000 38,044,000

1.83 2.44

0.29 0.48

a

Measurements for the calculation of the ratio Ukat/Upox were performed at the Wnal concentration of alcohol D 0.11 M.

The electronic spectrum of puriWed catalase-1 is shown in Fig. 2. From the absorbance at the two maxima at 405 nm (Soret peak) and 280 nm (protein maximum), a Rz value of 0.536 can be calculated (i.e., the ratio of absorbances between these two maxima). The  and 1, 2 bands at 628, 543, and 502 nm are also discernible as small peak and two shoulders, respectively. The observed Rz ratio is signiWcantly lower than the values for the majority of hydroperoxidases, and even smaller than the values found with most typical tetrameric catalases (typically around Rz D 1). Thus, the observed spectrum can be considered as atypical. Similar atypical spectra were observed also for some other catalases (e.g., from yeast—[16]). In most cases, however, low Rz values reXect partial loss of heme groups, so this possibility must also be considered with catalase-1 from C. terrigena pointing to a rather weak non-covalent binding of the prosthetic group in the apoprotein. The thermostability test demonstrates that this heme protein has typical features of enzymes isolated from mesophilic organisms and its stability rapidly decreases above 55 °C (Fig. 3A). Also the recorded pH optimum of catalase-1 (Fig. 3B) fairly matches the behavior of other typical catalases. This group of hydrogen peroxide

Fig. 2. Electronic spectrum of catalase-1 from Comamonas terrigena N3H puriWed to homogeneity. Besides the protein peak at 279 nm the typical maximum of the prosthetic heme group (Soret band) can be detected at 405 nm. Also shoulders at 502 and 543 nm and a small peak at 628 nm are discernible.

M. Zámocký et al. / Protein Expression and PuriWcation 36 (2004) 115–123

119

Fig. 3. Enzymatic properties of puriWed catalase-1 from Comamonas terrigena N3H. (A) Thermostability of the puriWed catalase-1 Samples were incubated at 30, 40, 50, 55, 60, and 65 °C and aliquots were drawn at the indicated times to perform the standard catalase assay described in Materials and methods. The activity is given as the percentage of control sample (incubated at room temperature). (B) pH dependence of the catalatic activity of puriWed catalase-1. Samples were incubated in buVers of the respective pH values at room temperature for 10 min. Afterwards aliquots were drawn to perform the spectrophotometric assay at 240 nm as described in Materials and methods.

degrading enzymes generally exhibits a broad pH optimum between pH 6 and 10 [5]. In our case, we can observe two distinct pH optima, but nearly optimal activity is observed also in the range between them. This closely resembles the situation reported for the catalase from goat lung [17]. It is interesting to compare the properties of catalase1 from C. terrigena with those of other evolutionary related catalases that were described before. Catalase from Comamonas compransoris [18] was isolated from bacteria not exposed to any kind of oxidative stress. The speciWc catalatic activity is much lower than for the enzyme described herein, and the authors did not give detailed characteristics of the puriWed enzyme. In the case of the catalase from Staphylococcus warneri [19], the

catalase was puriWed 310-fold but even this enzyme exhibited a speciWc activity of only 10,800 U/mg which is roughly Wve times lower than the high speciWc activity of our puriWed enzyme (Table 1). However, all three mentioned catalases have some characteristic features in common. This includes a Soret band maximum at 406 nm, and, above all, the native form of these monofunctional catalases appears to be a dimer of subunits, a feature that is very rarely present among these enzymes since the majority of bacterial catalases are more stable as tetramers in the native state. Until now, no sequence information was available for these types of catalases. The periplasmic catalase from Vibrio Wscheri [20] is induced by both oxidative stress and by mild starvation stress upon approaching the stationary phase of growth.

Fig. 4. Trace of the mass spectrometry analysis of catalase-1.

120

M. Zámocký et al. / Protein Expression and PuriWcation 36 (2004) 115–123

Unfortunately, no data on the speciWc catalatic activity of this catalase in puriWed form are given. The level of the activity in crude extracts is very similar to results obtained by us with Catalase-1 from C. terrigena. Mass spectrometry output and sequence comparison The total mass of a single subunit of the puriWed catalase (55,417 Da) is obvious from Fig. 4 and falls into the range that is typical for a large group of heme-containing bacterial catalases. Interestingly, also a small peak corresponding to the dimeric form is visible in Fig. 4 (112,774 Da). The bands at 27,000 and 14,000 Da can be attributed to proteolytic degradation products that are co-puriWed during the isolation procedure as shown in our previous work [21]. All mass fragments obtained after tryptic digest of the band corresponding to the native dimer isolated form the native gradient polyacrylamide gel are listed in Table 3. After inspection of these masses and the respective deduced sequences, it is possible to localize the peptides A, B, E, L, and M in the sequences of related bacterial catalases. The peptides E and L can be located on the distal side of the prosthetic heme group whereas peptides B and M are situated on the proximal side of the prosthetic heme group as supported by Blast searches. These two sequence stretches reXect the areas of the highest sequence homology of the entire catalase

molecule [3]. Additionally, peptide A was obtained by N-terminal Edman sequencing. All Wve located fragments can be used for the sequence analysis presented below and were already deposited in Swiss-Prot protein sequence database. Besides these Wve partial sequences there are 20 other sequenced peptides that cannot be located unambiguously in any known region of a typical catalase. Either they are too short or they are probably from less conserved regions. They will be located only after the corresponding DNA sequence will be translated in the entire protein sequence (work is currently underway). Phylogenetic relationship of highly conserved sequence regions of bacterial catalases Multiple sequence alignment of 34 related sequences belonging to the catalase family and similar to fragments A, B, E, L, and M of Catalase-1 from C. terrigena is presented in Fig. 5A. Abbreviations used in the alignment are listed in Table 4. Several typical sequence patterns of this widely spread protein family can be distinguished. The highly conserved areas corresponding to the distal and proximal side of the prosthetic heme group can be deduced from the consensus sequence and are located in the sequence of Comamonas terrigena as follows: the amino acid positions L1-E10 are located in an almost invariable region of the lower part of the

Table 3 Output of MALDI-TOF for tryptic fragments revealing sequence stretches of Comamonas terrigena N3H catalase-1 Peptide ID

m/z

Mr

Sequence

Considerations

A B C D E F G H I

— 999.48 521.28 534.30 1073.52 1086.45 548.30 595.83 647.87

1429.13 998.48 1040.49 1066.58 1072.53 1085.45 1094.64 1189.66 1293.69

N-terminal Edman sequencing Area of proximal ligand

J K L M

432.20 651.80 512.92 517.90

1293.71 1301.66 1535.73 1550.84

THCLTTAAGAPVA LFSYGDAA LTPDAQQ GVSEHLVQ GAADAERDIR SSHYATQH LLPASNE ARPEHLVQ LAGVSSLAQ or LAGMNLARNG or LAGVSEHLVQ AGVSEHLVQ or VAGVSEHLVQ NGFHNWFP FSTVAGERGAADAER LGVNHQHIPVNAPR

N O P Q R T U

527.27 793.90 532.61 849.80 599.30 1013.02 676.70

1578.84 1585.77 1595.75 1697.78 1794.92 2024.00 2027.10

V W X Y Z

548.76 731.35 581.29 803.40 918.10

2191.04 2191.05 2321.16 2407.11 2751.26

LLQDLWHLE FGTFTVTGDLT or FGTFTVTWVT SDLNAPEHLVQ or SDLNAQTS YGGTLAYEPNT VFSDLGKKTEVFA TAAKPVANNQDD AEPVANNKDSLTAGP or AKPVANNKDSLTKP NASNE or MFHENASNE HTYSFLNASNE QADPAYGQRAD FYWFQPGDLF or FYDAFQPGDLF PANLVPGLG or SFTPANLVPGLG

The distal side of heme, substrate channel

The distal side of heme, substrate channel The proximal side of heme not far from essential tyrosine

M. Zámocký et al. / Protein Expression and PuriWcation 36 (2004) 115–123

121

Fig. 5. Sequence analysis of catalase-1 from Comamonas terrigena. (A) Multiple sequence alignment of conserved regions on the distal and proximal side of the prosthetic heme group. Abbreviations of bacterial catalases are listed in Table 4. (B) The unrooted tree showing phylogenetic relationship of bacterial catalases deduced from the conserved regions presented in (A). Abbreviations are listed in Table 4.

substrate channel on the heme’s distal side. The overall sequence similarity in this region is 58%. On the other hand, residues B1-M14 are located in the highly conserved proximal side of the prosthetic heme group. Interestingly, this region reveals a nearly identical overall sequence similarity of 56%. Residue B5 in C. terrigena catalase-1 is most likely the proximal ligand of the heme iron. The corresponding position is fully conserved in all aligned catalases (Fig. 5A on the right). The rather high sequence identity in both essential regions allows us to investigate the phylogenetic relationship of catalase-1 from C. terrigena with other related heme catalases. An unrooted phylogenetic tree of 34 related typical catalases is presented in Fig. 5B. From this Wgure, it is obvious that the closest phylogenetic neighbor of catalase-1 is the catalase from the bacterium Vibrio Wscheri. Both these catalases are located on a small branch together with Nitrosomonas europaea catalase and can be classiWed as belonging to clade III of bacterial catalases [22,23]. It is also obvious

that this catalase is closely related also with several catalases from pathogenic microorganisms, e.g., from Hemeophilus inXuenzae, Neisseria meningitidis, B. pertusis, and Yersinia pestis. So the elucidation of the expression and properties of catalase-1 from C. terrigena can help us to judge the importance of this hydroperoxidase for the survival of mentioned pathogenic microorganisms.

Conclusions The intracellular catalase-1 puriWed from C. terrigena N3H is a monofunctional heme-containing catalase, which most likely forms stable dimers in its catalytically active form. It is induced after the contact of bacterial cells with various oxidative stressors. The puriWcation from the crude extract comprising the consecutive steps of hydrophobic, molecular sieve,

122

M. Zámocký et al. / Protein Expression and PuriWcation 36 (2004) 115–123

Fig. 5 (continued)

and ion exchange chromatography led to a 195-fold puriWed sample of this bacterial hydroperoxidase. We provide here the Wrst information on the sequence of conserved regions of this heme protein. The essential

residues responsible for the catalytic cycle are conserved in this monofunctional catalase. The closest known phylogenetic neighbor of catalase1 from C. terrigena is the catalase from Vibrio Wscheri.

M. Zámocký et al. / Protein Expression and PuriWcation 36 (2004) 115–123 Table 4 GenBank accession numbers of 33 catalases used for sequence comparison with Comamonas terrigena catalase-1 Abbreviation used GenBank Accession # Organism Aactinomyc Acalcoacet Babortus Bfragilis Bfungorum Bpertussis Bsubtilis Cjejuni Dvulgaris Halomonas HinXuenza Hpylori Lsake Mbarkeri Mmazei Neuropea Nmeningiti Nostocp Onchocerca PXuoresce Pgulae Pmirabilis Pmultocida Pputida Rhizobium Rhrubrum Rmetallidura Saureus Scoelicolo Smelioti Sviolaceus VWscheri Ypestis

AAF17882

Actinobacillus actinomycetemcomitans BAC55928 Acinetobacter calcoaceticus A55227 Brucella abortus P45737 Bacteroides fragilis NZ_AAAC01000245 Burkholderia fungorum P48062 Bordetella pertussis M80796 Bacillus subtilis NP_282531 Campylobacter jejuni BAA34670 Desulfovibrio vulgaris BAB88221 Halomonas sp. SK1 P44390 Haemophilus inXuenzae AAC16068 Helicobacter pylori P30265 Lactobacillus sakei O93662 Methanosarcina barkeri NP_634581 Methanosarcina mazei ZP_00003530 Nitrosomonas europea NP_282903 Neisseria meningitidis ZP_00112076 Nostoc punctiforme Q27710 endobacterium of Onchocerca volvulus P77924 Pseudomonas Xuorescens BAC20190 Porphyromonas gulae AJ400965 Proteus mirabilis NP_244969 Pasteurella multocida U63511 Pseudomonas putida U59271 Rhizobium meliloti NZ_AAAG01000015 Rhodospirillum rubrum ZP_00026543 Ralstonia metallidurans AP003133 Staphylococcus aureus NP_631632 Streptomyces coelicolor P95631 Sinorhizobium melioti X74791 Streptomyces violaceus AAC38344 Vibrio Wscheri NP_404811 Yersinia pestis

These sequences were obtained after sequence similarity searches of peptides A, B, E, and L of catalase-1 from Comamonas terrigena (Table 3) with Blast [15].

Acknowledgments Our investigations were supported by Vega Grant Nr.2/2056/22. References [1] J.G. Scandalios, The rise of ROS, Trends Biochem. Sci. 27 (2002) 483–486. [2] J. Basran, N. Bhanji, A. Basran, D. Nietlispach, S. Mistry, R. Meskys, N.S. Scruton, Mechanistic aspects of the covalent Xavoprotein dimethylglycine oxidase of Arthrobacter globiformis studied by stoppedXow spectrophotometry, Biochemistry 41 (2002) 4733–4743. [3] M. Zámocký, F. Koller, Understanding the structure and function of catalases: clues from molecular evolution and in vitro mutagenesis, Prog. Biophys. Mol. Biol. 72 (1999) 19–66.

123

[4] E.R. Rocha, T. Selby, J.P. Coleman, C.J. Smith, Oxidative stress response in an anaerobe Bacteroides fragilis: a role for catalase in protection against hydrogen peroxide, J. Bacteriol. 178 (1996) 6895–6903. [5] P. Nicholls, I. Fita, P.C. Loewen, Enzymology and structure of catalases, Adv. Inorg Chem. 51 (2001) 51–90. [6] E.J. Yun, Y.N. Lee, Production of two diVerent catalase-peroxidases by Deinococcus radiophilus, FEMS Microbiol. Lett. 184 (2000) 155–159. [7] M. Zámocký, B. Polek, J. Godobíková, F. Koller, Oxidative stressinduced expression of catalases in Comamonas terrigena, Folia Microbiol. 47 (2002) 335–340. [8] M. Proknová, J. Augustín, A. Vrbanová, Enrichment, isolation and characterization of dialkyl sulfosuccinate degrading bacteria Comamonas terrigena N3H and Comamonas terrigena N1C, Folia Microbiol. 42 (1997) 635–639. [9] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. [10] W. Woodbury, A.K. Spencer, M.A. Stahman, An improved procedure using ferricyanide for detecting catalase isozymes, Anal. Biochem. 44 (1971) 301–305. [11] R. Roggenkamp, H. Sahm, F. Wagner, Microbial assimilation of methanol and function of catalase in Candida boidinii. Microbial assimilation of methanol and function of catalase in Candida boidinii, FEBS Lett. 41 (1974) 283–286. [12] M. Zámocký, C. Herzog, L.M. Nykyri, F. Koller, Site-directed mutagenesis of the lower parts of yeast catalase A leads to highly increased peroxidatic activity, FEBS Lett. 367 (1995) 241–245. [13] F. Jeanmougin, J.D. Thompson, M. Gouy, M. Higgins, T.J. Gibson, Multiple sequence alignment with ClustalX, Trends Biochem. Sci. 23 (1998) 403–405. [14] S.F. Altschul, T.L. Madden, A.A. SchäVer, J. Zhang, Z. Zhang, W. Miller, D.J. Lipman, Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res. 25 (1997) 3389–3402. [15] S.G. Kalko, J.L. Gelpi, I. Fita, M. Orozco, Theoretical study of the mechanism of substrate recognition by catalase, J. Am. Chem. Soc. 123 (2001) 9665–9672. [16] T.C. Seah, A.R. Bhatti, J.G. Kaplan, A catalactic nucleoprotein of baker’s yeast, FEBS Lett. 85 (1978) 305–309. [17] U. Chatterjee, G.G. Sanwal, PuriWcation and characterization of catalase from goat (Capra capra) lung, Mol. Cell. Biochem. 126 (1993) 125–133. [18] D. Nies, H.G. Schlegel, Catalase from Comamonas compransoris, J. Gen. Appl. Microbiol. 28 (1982) 311–319. [19] K. Mizuno, M. Kahikara, M. Kohno, T.L. Ha, K. Sonomoto, A. Ishizaki, Catalase of Staphylococcus warneri ISK-1 isolated from Nukadoko, J. Fac. Agr. Kyushu Univ. 44 (2000) 329–338. [20] K.L. Visick, E.G. Ruby, The periplasmic, group III catalase of Vibrio Wscheri is required for normal symbiotic competence and is induced both by oxidative stress and by approach to the stationary phase, J. Bacteriol. 180 (1998) 2087–2092. [21] M. Zámocký, F. Koller, Homogenates of yeast cultures with engineered catalases F148V and V111A reveal higher speciWc activities after incubation at permissive temperature, Folia Microbiol. 42 (1997) 457–462. [22] G. Regelsberger, C. Jakopitsch, L. Plasser, H. Schwaiger, P.G. Furtmüller, G.A. Peschek, M. Zámocký, C. Obinger, Occurrence and biochemistry of hydroperoxidases in oxygenic phototropic prokaryotes (cyanobacteria), Plant Physiol. Biochem. 40 (2002) 479–490. [23] M.G. Klotz, G.R. Klassen, P.C. Loewen, Phylogenetic relationships among prokaryotic and eukaryotic catalases, Mol. Biol. Evol. 14 (1997) 951–958.