Characterization of Monofunctional Catalase KatA from Radioresistant Bacterium Deinococcus radiodurans
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JOURNAL OF BIOSCIENCE AND BIOENGINEERING © 2006, The Society for Biotechnology, Japan
Vol. 101, No. 4, 315–321. 2006
DOI: 10.1263/jbb.101.315
Characterization of Monofunctional Catalase KatA
from Radioresistant Bacterium Deinococcus radiodurans
Issei Kobayashi,1 Takashi Tamura,1 Haitham Sghaier,2,3 Issay Narumi,2
Shotaro Yamaguchi,4 Koichi Umeda,4 and Kenji Inagaki1*
Department of Biofunctional Chemistry, Graduate School of Natural Science and Technology, Okayama University,
1-1-1 Tsushima-naka, Okayama 700-8530, Japan,1 Gene Resource Research Group, Quantum Beam Science
Directorate, Japan Atomic Energy Agency, 1233 Watanuki, Takasaki, Gunma 370-1292, Japan,2
Department of Biological and Chemical Engineering, Faculty of Engineering, Gunma
University, 1-5-1 Tenjin, Kiryu, Gunma 376-8515, Japan,3 and Amano Enzyme Inc.,
Gifu R&D Center, 4-179-35 Sue-cho, Kakamigahara, Gifu 509-0108, Japan 4
Received 31 October 2005/Accepted 11 January 2006
Catalase plays a key role in protecting cells against toxic reactive oxygen species. Here we re-
port on the cloning, purification and characterization of a catalase (KatA, DR1998) from the ex-
tremely radioresistant bacterium Deinococcus radiodurans. The size of purified D. radiodurans
KatA monomer was 65 kDa while gel filtration revealed that the size of the enzyme was 240 kDa,
suggesting that KatA formed a homotetramer in solution. Purified KatA displayed a final specific
activity of 68,800 U/mg of protein. The catalase activity of KatA was inhibited by sodium azide,
sodium cyanide and 3-amino-1,2,4-triazole. The absorption spectrum of KatA exhibited a Soret
band at 408 nm. The position of the spectral peak remained unchanged following reduction of
KatA with dithionite. No peroxidase activity was found for KatA. These results demonstrate that
D. radiodurans KatA is a typical monofunctional heme-containing catalase. The stability of KatA
with respect to H2O2 stress was superior to that of commercially available Aspergillus niger and
bovine liver catalases. The relative abundance of KatA in cells in addition to the H2O2 resistance
property may play a role in the survival strategy of D. radiodurans against oxidative damage.
[Key words: antioxidant, catalase, Deinococcus radiodurans, gene expression, hydrogen peroxide resistance]
Hydrogen peroxide (H2O2) and other reactive oxygen over 100 genomic DNA double-strand breaks, the primary
species (ROS) such as superoxide anion (O2–) are produced lethal lesions resulting from exposure to ionizing radiation.
in aerobes from the partial reduction of molecular oxygen Although this resistance has been attributed to its highly
(1). In the Fenton reaction, H2O2 readily reacts with avail- condensed nucleoid structure together with its effective
able transition metals such as ferrous iron to form the hy- DNA repair capacity (5–8), some evidence suggests that
droxyl radical (OH•), a highly reactive and toxic oxidant (2). radiation resistance in D. radiodurans involves the use of
ROS including H2O2 and O2– are also generated during the scavenging systems against ROS. Basal levels of catalase
radiolysis of water, an indirect effect of ionizing radiation and SOD activities in cell extracts from an exponential
(3). Irrespective of the source, cells become vulnerable to growing culture of D. radiodurans were shown to be over
damage, a condition referred to as oxidative stress, when 120-fold and 3-fold greater, respectively, compared with
ROS exceed the capacity of endogenous scavengers to elim- Escherichia coli (9, 10). D. radiodurans cultures pretreated
inate them (2). As a defense against ROS, organisms con- with 10 mM H2O2 were more resistant to the lethal effect of
tain two major antioxidant enzymes; superoxide dismutase γ irradiation, and was accompanied with a 2.4-fold increase
(EC 1.15.1.1) which catalyzes the disproportionation of O2– in total catalase activity that was probably due to induction
to H2O2 and oxygen, and catalase (EC 1.11.1.6) which cata- of a catalase protein (10). Activity staining on a nondenatur-
lyzes the disproportionation of H2O2 to water and oxygen ing polyacrylamide gel revealed that D. radiodurans wild-
(2). type strain possessed activity corresponding to two cata-
Deinococcus radiodurans, an aerobic, nonmotile and non- lases and one SOD (11). Disruption of the D. radiodurans
sporing eubacterium, is characterized by its extreme resis- katA and sodA genes resulted in complete loss of the major
tance to both lethal and mutagenic effects of a variety of catalase and total SOD activity, respectively. Both disrup-
DNA-damaging agents, and possesses an unusually high re- tants exhibited greater sensitivity to ionizing radiation at
sistance to ionizing radiation (4). This bacterium can mend high doses (11). Defect in the pprI gene, which encodes a
novel regulatory protein responsible for extreme radioresis-
* Corresponding author. e-mail: kinagaki@cc.okayama-u.ac.jp tance in D. radiodurans, resulted in hypersensitivity to radi-
phone/fax: +81-(0)86-251-8299 ation and was accompanied with reduced catalase activity
315316 KOBAYASHI ET AL. J. BIOSCI. BIOENG.,
(12). Overexpression of the D. radiodurans recX gene, which was stopped by the addition of 100 µg/ml of bovine liver catalase
encodes a negative regulator for recombinase A (RecA), to the culture. The number of colony-forming units was determined
suppressed radioresistance and catalase activity (13, 14). by plating serial dilutions of the culture onto TY agar plates fol-
These results suggest that catalase activity is involved in the lowed by counting after 24 h growth at 37°C.
Protein purification E. coli strain JM109 carrying pMH11
radioresistance of this bacterium.
(1 l) was cultured at 30°C in LB broth containing 50 µg/ml of am-
Although the purification and characterization of a pro- picillin. When the optical density at 600 nm reached approximately
tein with SOD activity from D. radiodurans has been re- 0.5, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a
ported (15), the biochemical property of the D. radiodurans final concentration of 1 mM. Growth was continued for an addi-
catalase had not yet been investigated. Since catalases, tional 4 h, after which time, cells were harvested by centrifugation
together with SOD, play a key role in protecting cells and then washed with Buffer S consisting of 10 mM potassium
against toxic ROS, here we report on the cloning, purifi- phosphate buffer (pH 7) and 0.01% 2-mercaptoethanol. The cell
cation and characterization of a catalase (KatA) from D. pellet was stored at −20°C until further use.
radiodurans. All subsequent steps were performed at 4°C. The cell pellet
(5 g) was resuspended in Buffer S, disrupted by sonication (120 W,
15 min), centrifuged at 37,000×g for 60 min to remove cell debris,
and the supernatant (20 ml) subsequently applied to a DEAE-
MATERIALS AND METHODS
Toyopearl column (2.6 × 10 cm; Tosoh, Tokyo) equilibrated with
Bacterial strains and growth conditions D. radiodurans Buffer S. The column was washed with Buffer S containing 100
strain KR1 (16) was grown at 30°C in TGY broth containing mM NaCl and then eluted with Buffer S containing 150 mM NaCl
0.5% (wt/vol) tryptone–peptone, 0.1% (wt/vol) glucose and 0.3% at a flow rate of 2 ml/min. Fractions containing high catalase activ-
(wt/vol) yeast extract. E. coli strain JM109 (Toyobo, Osaka) was ity were pooled, concentrated to 15 ml at 4000×g using an Amicon
grown in Luria–Bertani (LB) broth containing 1% (wt/vol) tryp- Ultra-15 centrifugal filter device (Millipore, Billerica, MA, USA),
tone, 0.5% (wt/vol) yeast extract and 0.5% NaCl, or tryptone yeast and then applied to a Ceramic Hydroxyapatite Type-1 column
extract (TY) broth containing 1% (wt/vol) tryptone, 0.5% (wt/vol) (2.6× 3 cm; Bio-Rad Laboratories, Hercules, CA, USA) equilibrated
yeast extract and 0.8% NaCl. Solid media contained 1.5% (wt/vol) with 10 mM potassium phosphate buffer (pH 7). The column was
agar. Fifty micrograms of ampicillin per ml was supplemented in eluted with a linear gradient of 10 to 100 mM potassium phosphate
the medium when necessary. buffer (pH 7) at a flow rate of 1.5 ml/min. Fractions correspond-
Construction of expression plasmid pMH11 D. radiodurans ing to peak catalase activity were pooled, concentrated to 8 ml at
genomic DNA was isolated as previously described (16). Briefly, 8000×g using an Amicon YM10 unit (Millipore) and then applied
D. radiodurans cells from 24-h culture were harvested, washed in to a Sephacryl S-300 HR column (2.6× 100 cm; GE Healthcare
NE solution containing 0.15 M NaCl and 0.1 M EDTA (pH 8), in Bio-Science Corp., Piscataway, NJ, USA) equilibrated with Buffer
butanol-saturated NE solution to facilitate the lysis of the cell walls, S containing 150 mM NaCl. The column was eluted with the same
and again in NE solution. The washed cells were incubated in NE buffer at a flow rate of 1 ml/min and fractions corresponding to
solution containing 10 mg/ml of lysozyme (Sigma, St. Louis, MO, peak catalase activity were pooled. Following concentration using
USA) at 37°C for 1 h. Subsequently, the genomic DNA was iso- an Amicon YM10 unit, the pooled fractions (5 ml) were applied to
lated and purified by the method of Saito and Miura (17). A set of a Ceramic Hydroxyapatite Type-1 column (2.6 ×3 cm) equilibrated
20-mer mixed oligonucleotide probes, 5′-GA(C/T)GA(A/G)AA with 10 mM potassium phosphate buffer (pH 7). The column was
(C/T)AA(C/T)AA(A/G)GG(G/C)GT-3′, was used to clone the washed with 50 mM potassium phosphate buffer (pH 7) and then
katA gene from D. radiodurans genomic DNA. Southern blot hy- eluted with 100 mM potassium phosphate buffer (pH 7) at a flow
bridization was performed as previously described (16). Briefly, rate of 1.5 ml/min. Fractions containing high catalase activity were
the mixed oligonucleotide probes were labeled with digoxigenin collected, concentrated to 2 ml using an Amicon YM10 unit and
using a DIG-ULS labeling kit (Kreatech Diagnostics, Amsterdam, then used for the subsequent biochemical experiments.
Netherlands). DNA fragments after electrophoresis were trans- Enzyme assay and protein concentration determination
ferred from agarose gels to positively charged nylon membrane Catalase activity was determined spectrophotometrically by moni-
(Roche, Basel, Switzerland) with alkaline solution (0.4 N NaOH, toring the decrease in absorbance at 240 nm resulting from the dis-
1 M NaCl) for 24 h. DNA hybridization signals were detected as appearance of H2O2 (ε240 =43.6 M–1 cm–1) (18). One unit (U) of
recommended by the supplier after stringency washing (two suc- catalase activity was defined as that activity required to destroy
cessive washes at room temperature for 5 min in 30 mM sodium ci- 1 µmol H2O2 per min. Aspergillus niger catalase and bovine liver
trate plus 0.3 M NaCl [2 ×SSC] and 0.1% sodium dodecyl sulfate catalase were obtained from Amano Enzyme (Nagoya) and Sigma,
[SDS], followed by two successive washes at 68°C for 15 min in respectively, and used as references. The peroxidase activity of the
0.5 ×SSC and 0.1% SDS). A 5.3-kb NspI fragment containing the catalase was examined by measuring the rate of oxidation of 0.25
katA gene was inserted into the SphI site of pUC19 (Takara Bio, mM o-dianisidine (ε460= 11.3 mM–1 cm–1) in the presence of 10
Otsu). The resulting plasmid, pKDR1, was digested with SmaI and mM H2O2 (18). Reactions were monitored spectrophotometrically
then subcloned into the HincII site of pUC118 (Takara Bio), yield- at 460 nm. The protein concentration was determined using a Bio-
ing plasmid pMH1. In an effort to position the katA gene under Rad Protein Assay kit with bovine serum albumin as the standard.
control of the lacZ promoter, pMH1 was digested with KpnI and All assays were carried out at room temperature unless otherwise
NcoI, blunted with T4 DNA polymerase (Takara Bio) and then self- indicated.
ligated, yielding the expression plasmid pMH11. DNA sequencing
was performed using an ABI PRISM 377 DNA Sequencer (Applied
Biosystems, Foster City, CA, USA). RESULTS
E. coli H2O2 survival assay E. coli preculture grown at 30°C
in LB broth was inoculated in fresh LB broth to an initial OD600 of Cloning of the D. radiodurans katA gene In an effort
0.1. Thirty percent H2O2 was added to a final concentration of to clone the D. radiodurans katA gene, the sequence com-
100 mM and the sample was incubated at room temperature. Fol- posed of DENNKGV, the N-terminal amino acid sequence
lowing incubation for the indicated amount of time, the reaction of the catalase (11, 19), was used to design a set of mixedVOL. 101, 2006 CHARACTERIZATION OF D. RADIODURANS CATALASE 317
FIG. 2. SDS–PAGE analysis of the expression of D. radiodurans
KatA in E. coli JM109 cells and its purification. Each protein sample
FIG. 1. Survival of exponential phase cells of E. coli JM109 (open (2 to 3 µg) was mixed with an equal volume of loading buffer contain-
circles) and E. coli JM109 carrying plasmid pMH11 (closed circles) ing 2% SDS and 5% 2-mercaptoethanol, and heated at 100°C for
following exposure to 100 mM H2O2. Data represents the mean of at 10 min prior to being resolved in a 10% SDS–polyacrylamide gel. The
least three independent experiments. resolved proteins in the gel were stained with Coomassie Brilliant
Blue R-250. The position of the 65-kDa KatA band is indicated on the
right. Lane 1, Molecular markers; lane 2, crude protein extract from
oligonucleotide probes for Southern blot analysis (see sonicated cells; lane 3, pooled fractions following DEAE-Toyopearl
Materials and Methods). Southern blot analysis showed that chromatography; lane 4, pooled fractions following 1st Hydroxyapa-
the probes hybridized to a single 5.3-kb NspI fragment de- tite chromatography; lane 5, pooled fractions following Sephacryl
rived from D. radiodurans genomic DNA. This fragment S-300 chromatography; lane 6, pooled fractions following 2nd Hy-
droxyapatite chromatography.
was cloned into the SphI site of pUC19, yielding plasmid
pKDR1. DNA sequencing analysis revealed that the se-
quence of the 5.3-kb NspI region was identical to the corre- SDS–PAGE analysis indicated the presence of a major pro-
sponding region of the published D. radiodurans genome tein band of approximately 65 kilodaltons (kDa) (Fig. 2).
sequence (20), and that it contained the entire katA gene This is in good agreement with the molecular weight
(DR1998). In order to position the katA gene under control (60,512 Da) deduced from the amino acid sequence of the
of the lacZ promoter, a 2.6-kb SmaI fragment of pKDR1 D. radiodurans katA gene product (20). KatA was purified
was subcloned into pUC118, digested with KpnI and NcoI, to greater than 95% purity as determined by SDS–PAGE.
blunted with T4 DNA polymerase and then self-ligated. The Purified KatA displayed a high final specific activity of
resulting expression plasmid, pMH11, was introduced into 68,800 U/mg of protein at 30°C. No peroxidase activity was
E. coli strain JM109. found for KatA using the o-dianisidine method. The native
The E. coli transformant carrying pMH11 grew on a TY molecular mass of KatA was estimated to be 240 kDa by gel
agar plate containing 10 mM H2O2, whereas E. coli strain filtration using Sephacryl S-300 HR (data not shown). This
JM109 without pMH11 did not (data not shown). Figure 1 suggests that purified KatA is composed of four identical
shows the survival of the transformant following exposure subunits.
to 100 mM H2O2. The pMH11 transformant exhibited higher Effect of inhibitors on activity and spectroscopic prop-
resistance to H2O2 compared with E. coli without the plas- erties of the catalase Catalase activity was strongly in-
mid. This suggests that the pMH11 transformant produced hibited by sodium azide and sodium cyanide, inhibitors of
functional KatA protein (a product of the D. radiodurans heme-containing catalase (Table 2). Treatment of D. radio-
katA gene) even without IPTG induction. durans KatA with 3-amino-1,2,4-triazole at 0.1 mM and 1.0
Purification of D. radiodurans KatA Purification of mM resulted in marginal reduction of catalase activity. As
D. radiodurans KatA generated following induction with shown in Fig. 3, the absorption spectrum of D. radiodurans
IPTG in E. coli carrying pMH11 was accomplished by a KatA exhibited a Soret band at 408 nm typical of a heme-
four-step chromatographic procedure utilizing DEAE- containing catalase (21). This peak shifted to 422 nm fol-
Toyopearl, 1st Hydroxyapatite, Sephacryl S-300 HR and lowing the addition of 10 mM sodium cyanide, indicating
2nd Hydroxyapatite columns (Table 1). The procedure re- direct binding of cyanide to the heme moiety. The position
sulted in approximately 21-fold purification with 41% yield. of the spectral peak remained unchanged following treat-
TABLE 1. Summary of the purification of D. radiodurans catalase KatA from overexpressed E. coli cells
Total protein Total activity Specific activity Yield Purification
Fraction
(mg) (U) (U/mg) (%) (fold)
Crude extract 609 201 ×104 3300 100 1.0
DEAE-Toyopearl 86 204 ×104 23700 101 7.2
1st hydroxyapatite 51 180 ×104 35300 90 10.7
Sephacryl S-300 HR 26 176 ×104 67700 87 20.5
2nd hydroxyapatite 12 82.5 ×104 68800 41 20.8
One unit (U) of activity is defined as that activity required to destroy 1 µmol H2O2 per min.318 KOBAYASHI ET AL. J. BIOSCI. BIOENG.,
TABLE 2. Effect of inhibitors on D. radiodurans KatA activity
Final concentration
Inhibitor % inhibitiona
(mM)
Sodium azide 0.1 93
1.0 99
Sodium cyanide 0.1 63
1.0 87
3-Amino-1,2,4-triazole 0.1 4
1.0 11
a
Purified protein (1 mg/ml) was treated with 0.1 mM or 1.0 mM in-
hibitor in Buffer S at room temperature for 30 min prior to the mea-
surement of catalase activity.
ment of the enzyme with 1 mM sodium dithionite. FIG. 3. Changes in the absorption spectrum of purified D. radio-
Effect of temperature on catalase activity Catalase durans KatA following cyanide or dithionite treatment.
activity was assayed at various temperatures using purified
KatA and was compared with that of commercially avail-
able bovine liver and A. niger catalases (Fig. 4A). Purified
KatA was active over a temperature range from 20°C to
70°C, with optimum activity occurring at 30°C. The opti-
mum assay temperature demonstrating the enzymatic activ-
ity of bovine liver and A. niger catalases was 40°C and
50°C to 60°C, respectively. At 70°C, the activity of A. niger,
bovine liver and D. radiodurans catalases was reduced to
82%, 45% and 20%, respectively, of each activity at the op-
timum assay temperature.
The stability of D. radiodurans KatA to heat was exam-
ined by incubating the purified enzyme at a fixed tempera-
ture for 30 min (Fig. 4B). KatA retained 100% of its initial
activity following incubation at 40°C, and 82% of its activ-
ity following incubation at 50°C. The activity of KatA de-
creased significantly to 30% of the initial activity following
incubation at 60°C. Bovine liver catalase was less stable at
temperatures greater than 40°C and was completely unsta-
ble at 60°C. A. niger catalase activity was abolished follow-
ing incubation at 80°C. These results suggest that D. radio-
durans KatA is less heat-labile than bovine liver catalase
but more heat-labile than A. niger catalase.
Response to exogenously added H2O2 Many cata-
lases are known to be inhibited or inactivated by the sub-
strate H2O2 (22). The susceptibility of D. radiodurans KatA
to H2O2 stress was investigated by the dialysis method as
described in Materials and Methods. As shown in Fig. 5, the
residual activity of KatA decreased gradually with increas-
ing H2O2 concentration, demonstrating apparent substrate FIG. 4. (A) Effect of temperature on catalases from D. radio-
inhibition/inactivation of the enzyme. However, D. radiodu- durans, A. niger and bovine liver. Catalase activity was measured as
described in Materials and Methods at the temperatures indicated.
rans KatA retained 82% activity following exposure to 100 (B) Thermostability of catalases from D. radiodurans, A. niger and bo-
mM H2O2, whereas A. niger and bovine liver catalases re- vine liver. Enzymes were incubated for 30 min at the indicated temper-
tained 70% and 48% activity, respectively. This indicates atures prior to initiation of the reaction. Catalase activity was assayed
that the stability of D. radiodurans KatA to H2O2 stress was at room temperature. Symbols: squares, D. radiodurans KatA; circles,
superior to that of A. niger and bovine liver catalases under A. niger catalase; triangles, bovine liver catalase. Data represents the
mean of at least three independent experiments.
the experimental conditions used.
DISCUSSION alases exist as a homotetramer containing four protoheme
IX prosthetic groups per tetramer, with a molecular mass of
Catalases are classified into three general classes based 225 to 270 kDa. These enzymes are specifically inhibited by
upon structure and properties, being the monofunctional sodium cyanide and 3-amino-1,2,4-triazole, and are resis-
catalases, the bifunctional catalases that possess both cata- tant to reduction by sodium dithionite (24).
lase and peroxidase activity, and the non-heme or manga- In this study, we purified and characterized the catalase
nese-containing catalases (23). Typical monofunctional cat- KatA from D. radiodurans. The size of purified KatA wasVOL. 101, 2006 CHARACTERIZATION OF D. RADIODURANS CATALASE 319
subtilis than those of the B. subtilis vegetative catalases,
KatA and KatE (Fig. 6). B. subtilis KatX has no role in dor-
mant spore resistance to H2O2, but functions in protecting
germinating spores from H2O2 (31). Although D. radio-
durans does not form spore, the high similarity of the D.
radiodurans KatA to the B. subtilis KatX will provide a clue
to clarify the structure-function relationship of these en-
zymes.
Karlin and Mrázek (32) devised an algorithm to estimate
the protein abundance in a cell population on the basis of
codon usage, and applied the algorithm to predict the gene
expression levels of the D. radiodurans genome. Not unex-
pectedly, according to this prediction D. radiodurans KatA
was one of a number of highly-expressed proteins (32). The
FIG. 5. Effect of H2O2 on catalase activity. One hundred micro- proteomic analysis of D. radiodurans confirmed the pres-
liters of purified protein at a concentration of 1 mg/ml was dialyzed ence of KatA under most culture conditions tested, suggest-
against 0 to 100 mM H2O2 in Buffer S at 20°C for 10 min prior to the ing constitutive high expression (33). Thus, the abundance
measurement of catalase activity. Symbols: squares, D. radiodurans
KatA; circles, A. niger catalase; triangles, bovine liver catalase. Data of KatA in cells in addition to its H2O2 resistance might play
represents the mean of at least three independent experiments. a role in the survival strategy of D. radiodurans against oxi-
dative damage.
65 kDa by SDS–PAGE analysis (Fig. 2) and 240 kDa by gel Based on experiments showing that dehydration induces
filtration, suggesting that KatA forms a homotetramer in so- DNA double-strand breaks in D. radiodurans and that ion-
lution. KatA catalase activity was inhibited by sodium azide, izing radiation-sensitive mutants of this bacterium might
sodium cyanide and 3-amino-1,2,4-triazole, traditional in- also be sensitive to desiccation, Battista and his colleague
hibitors of heme-containing catalase (Table 2). The absorp- hypothesized that the ionizing radiation resistance pheno-
tion spectrum of KatA exhibited a Soret band at 408 nm. type of D. radiodurans might be a fortuitous consequence
The position of the spectral peak remained unchanged fol- of an evolutionary process that permitted this bacterium to
lowing reduction of KatA with dithionite (Fig. 3). Further- cope with commonly encountered environmental stressors
more, no peroxidase activity was found for KatA. These re- such as desiccation (desiccation adaptation hypothesis) (34).
sults demonstrate that D. radiodurans KatA is a typical Recently, the ionizing radiation-resistant fractions of two
monofunctional heme-containing catalase. Soung and Lee soil bacterial communities were investigated by exposing an
(25) showed that the catalases from wild-type D. radiodu- arid soil from desert area and a nonarid soil from forest area
rans (ATCC13939) possessed peroxidase activity as deter- to ionizing radiation (35). The authors of the aforemen-
mined by activity staining analysis. We assume that Soung tioned investigation argued that recovery of large numbers
and Lee detected false positive signals for peroxidase activ- of ionizing radiation-resistant bacteria including new spe-
ity. In this study, strain KR1 was used, another wild-type iso- cies of the genus Deinococcus from arid soil but not from
late of D. radiodurans. Therefore, the reason for this dis- nonarid soil provides ecological support for the desiccation
crepancy might have to do with strain polymorphism since adaptation hypothesis. The mechanism that mediates the
wild-type D. radiodurans isolates have been shown to ex- generation of ROS during desiccation remains poorly un-
hibit a wide variety of differences (26–29). derstood (36). ROS might be involved in the response of
It has been shown that D. radiodurans exhibits relatively arid soil bacteria to solar UV irradiation (37). D. radio-
high catalase activity (10). The specific activity of purified durans exhibits high resistance to UV light (4, 10). The rel-
D. radiodurans KatA protein was 68,800 U/mg (Table 1). ative abundance and superior resistance to H2O2 of catalase
This is not particularly outstanding when compared with the in D. radiodurans can be rationalized in terms of protection
catalase activity of other bacterial species. For example, it against solar UV-induced oxidative stress. However, it is not
was shown that the specific activity of purified catalase from necessarily the case for all members of the genus Deinococ-
the facultatively psychrophilic bacterium Vibrio rumoiensis cus, since two species (D. hohokamensis and D. navajonen-
was approximately 400,000 U/mg (30). However, in this sis) isolated from desert soil from Arizona lack catalase ac-
study we showed that, even under non-induced conditions, tivity (35). The optimum temperature required for activity
E. coli JM109 cells carrying the expression plasmid pMH11 of D. radiodurans KatA was 30°C (Fig. 4A), which is in
exhibited higher resistance to H2O2 compared with E. coli agreement with the optimum growth temperature for this
without the plasmid (Fig. 1). Moreover, purified D. radio- bacterium. D. radiodurans KatA was found not to be partic-
durans KatA protein was more stable to H2O2 stress than ularly thermostable (Fig. 4B). These results suggest that
commercially available A. niger and bovine liver catalases there is no significant relationship between high UV resis-
(Fig. 5). These results indicate that the D. radiodurans KatA tance and protein thermostability in this bacterium. This
possesses relatively superior ability in H2O2 resistance com- might reflect the evolution of D. radiodurans in the natural
pared to catalases from other species. In this context, it is habitat. Further comparative study using catalases from
worth noting that the deduced amino acid sequence of the other Deinococcus species will help to delineate the physio-
D. radiodurans KatA exhibited higher similarity to that of logical role of deinococcal catalases in oxidative stress pro-
KatX, which is the major catalase in dormant spores of B. tection.320 KOBAYASHI ET AL. J. BIOSCI. BIOENG.,
FIG. 6. Multiple amino acid sequence alignment of D. radiodurans KatA (DraKatA. seq), B. subtilis KatX (BsuKatX; UniProt accession
no. P94377), B. subtilis KatA (BsuKatA; UniProt accession no. P26901), and B. subtilis KatE (BsuKatE; UniProt accession no. P42234). Dots in-
dicate identical residues with D. radiodurans KatA. Dashes indicate gaps in the alignment. Numbers are the coordinates of each protein. Reverse
letters presents conserved residues.
ACKNOWLEDGMENTS REFERENCES
We thank A.B.M. Mamun Hossain for constructing plasmids 1. Fridovich, I.: The biology of oxygen radicals. Science, 201,
pKDR1, pMH1 and pMH11. We are also grateful to Dr. Akihiko 875–880 (1978).
Yamagishi for helpful discussions. 2. Imlay, J. A.: Pathways of oxidative damage. Annu. Rev.
Microbiol., 57, 395–418 (2003).
3. Jonah, C. D.: A short history of the radiation chemistry of
water. Radiat. Res., 144, 141–147 (1995).
4. Battista, J. R. and Rainey, F. A.: Family I. Deinococcaceae,
p. 395–403. In Boone, D. R. and Castenholz, R. W. (ed.),VOL. 101, 2006 CHARACTERIZATION OF D. RADIODURANS CATALASE 321
Bergey’s manual of systematic bacteriology, 2nd ed., vol. 1. datic activities. J. Biol. Chem., 254, 4245–4252 (1979).
Springer-Verlag, New York (2001). 22. Jones, P. and Suggett, A.: The catalase–hydrogen peroxide
5. Battista, J. R., Earl, A. M., and Park, M. J.: Why is Deino- system. Kinetics of catalatic action at high substrate concen-
coccus radiodurans so resistant to ionizing radiation? Trends trations. Biochem J., 110, 617–620 (1968).
Microbiol., 7, 362–365 (1999). 23. Chelikani, P., Fita, I., and Loewen, P. C.: Diversity of struc-
6. Narumi, I.: Unlocking radiation resistance mechanisms: still tures and properties among catalases. Cell. Mol. Life Sci., 61,
a long way to go. Trends Microbiol., 11, 422–425 (2003). 192–208 (2004).
7. Englander, J., Klein, E., Brumfeld, V., Sharma, A. K., 24. Nadler, V., Goldberg, I., and Hochman, A.: Comparative
Doherty, A. J., and Minsky, A.: DNA toroids: framework study of bacterial catalases. Biochim. Biophys. Acta, 882,
for DNA repair in Deinococcus radiodurans and in germinat- 234–241 (1986).
ing bacterial spores. J. Bacteriol., 186, 5973–5977 (2004). 25. Soung, N. K. and Lee, Y. N.: Iso-catalase profiles of Deino-
8. Zimmerman, J. M. and Battista, J. R.: A ring-like nucleoid coccus spp. J. Biochem. Mol. Biol., 33, 412–416 (2000).
is not necessary for radioresistance in the Deinococcaceae. 26. Southworth, M. W. and Perler, F. B.: Protein splicing of the
BMC Microbiol., 5, 17 (2005). Deinococcus radiodurans strain R1 Snf2 intein. J. Bacteriol.,
9. Yoshinaka, T., Yano, K., and Yamaguchi, H.: Cell lysis and 184, 6387–6388 (2002).
superoxide dismutase activities of highly radioresistant bac- 27. Islam, S. M., Hua, Y., Ohba, H., Satoh, K., Kikuchi, M.,
teria. Agric. Biol. Chem., 40, 227–229 (1976). Yanagisawa, T., and Narumi, I.: Characterization and distri-
10. Wang, P. and Schellhorn, H. E.: Induction of resistance to bution of IS8301 in the radioresistant bacterium Deinococcus
hydrogen peroxide and radiation in Deinococcus radiodurans. radiodurans. Gene Genet. Syst., 78, 319–327 (2003).
Can. J. Microbiol., 41, 170–176 (1995). 28. Eggington, J. M., Haruta, N., Wood, E. A., and Cox, M. M.:
11. Markillie, L. M., Varnum, S. M., Hradecky, P., and Wong, The single-stranded DNA-binding protein of Deinococcus
K. K.: Targeted mutagenesis by duplication insertion in the radiodurans. BMC Microbiol., 4, 2 (2004).
radioresistant bacterium Deinococcus radiodurans: radiation 29. Mennecier, S., Coste, G., Servant, P., Bailone, A., and
sensitivities of catalase (katA) and superoxide dismutase Sommer, S.: Mismatch repair ensures fidelity of replication
(sodA) mutants. J. Bacteriol., 181, 666–669 (1999). and recombination in the radioresistant organism Deinococ-
12. Hua, Y., Narumi, I., Gao, G., Tian, B., Satoh, K., Kitayama, cus radiodurans. Mol. Genet. Genomics, 272, 460–469
S., and Shen, B.: PprI: a general switch responsible for ex- (2004).
treme radioresistance of Deinococcus radiodurans. Biochem. 30. Yumoto, I., Ichihashi, D., Iwata, H., Istokovics, A., Ichise,
Biophys. Res. Commun., 306, 354–360 (2003). N., Matsuyama, H., Okuyama, H., and Kawasaki, K.:
13. Sheng, D., Gao, G., Tian, B., Xu, Z., Zheng, Z., and Hua, Purification and characterization of a catalase from the facul-
Y.: RecX is involved in antioxidant mechanisms of the radio- tatively psychrophilic bacterium Vibrio rumoiensis S-1T ex-
resistant bacterium Deinococcus radiodurans. FEMS Micro- hibiting high catalase activity. J. Bacteriol., 182, 1903–1909
biol. Lett., 244, 251–257 (2005). (2000).
14. Sheng, D., Liu, R., Xu, Z., Singh, P., Shen, B., and Hua, Y.: 31. Bagyan, I., Casillas-Martinez, L., and Setlow, P.: The katX
Dual negative regulatory mechanisms of RecX on RecA func- gene, which codes for the catalase in spores of Bacillus subti-
tions in radiation resistance, DNA recombination and conse- lis, is a forespore-specific gene controlled by σF, and KatX is
quent genome instability in Deinococcus radiodurans. DNA essential for hydrogen peroxide resistance of the germinating
Repair, 4, 671–678 (2005). spore. J. Bacteriol., 180, 2057–2062 (1998).
15. Juan, J. Y., Keeney, S. N., and Gregory, E. M.: Reconstitu- 32. Karlin, S. and Mrázek, J.: Predicted highly expressed and
tion of the Deinococcus radiodurans aposuperoxide dismu- putative alien genes of Deinococcus radiodurans and impli-
tase. Arch. Biochem. Biophys., 286, 257–263 (1999). cations for resistance to ionizing radiation damage. Proc. Natl.
16. Narumi, I., Cherdchu, K., Kitayama, S., and Watanabe, Acad. Sci. USA, 98, 5240–5245 (2001).
H.: The Deinococcus radiodurans uvrA gene: identification 33. Lipton, M. S., Pasa-Tolić, L., Anderson, G. A., Anderson,
of mutation sites in two mitomycin-sensitive strains and the D. J., Auberry, D. L., Battista, J. R., Daly, M. J.,
first discovery of insertion sequence element from deinobac- Fredrickson, J., Hixson, K. K., Kostandarithes, H., and
teria. Gene, 198, 115–126 (1997). other 12 authors: Global analysis of the Deinococcus radio-
17. Saito, H. and Miura, K.: Preparation of transforming de- durans proteome by using accurate mass tags. Proc. Natl.
oxyribonucleic acid by phenol treatment. Biochim. Biophys. Acad. Sci. USA, 99, 11049–11054 (2002).
Acta, 72, 619–629 (1963). 34. Mattimore, V. and Battista, J. R.: Radioresistance of Deino-
18. Ro, Y. T., Lee, H. I., Kim, E. J., Koo, J. H., Kim, E., and coccus radiodurans: functions necessary to survive ionizing
Kim, Y. M.: Purification, characterization, and physiological radiation are also necessary to survive prolonged desiccation.
response of a catalase-peroxidase in Mycobacterium sp. strain J. Bacteriol., 178, 633–637 (1996).
JC1 DSM 3803 grown on methanol. FEMS Microbiol. Lett., 35. Rainey, F. A., Ray, K., Ferreira, M., Gatz, B. Z., Nobre,
226, 397–403 (2003). M. F., Bagaley, D., Rash, B. A., Park, M. J., Earl, A. M.,
19. Tanaka, A., Hirano, H., Kikuchi, M., Kitayama, S., and Shank, N. C., and other 5 authors: Extensive diversity of
Watanabe, H.: Changes in cellular proteins of Deinococcus ionizing-radiation-resistant bacteria recovered from Sonoran
radiodurans following γ-irradiation. Radiat. Environ. Biophys., Desert soil and description of nine new species of the genus
35, 95–99 (1996). Deinococcus obtained from a single soil sample. Appl. Envi-
20. White, O., Eisen, J. A., Heidelberg, J. F., Hickey, E. K., ron. Microbiol., 71, 5225–5235 (2005).
Peterson, J. D., Dodson, R. J., Haft, D. H., Gwinn, M. L., 36. Leprince, O., Hendry, G. A. F., Atherton, N. M., and
Nelson, W. C., Richardson, D. L., and other 22 authors: Waters-Vertucci, C.: Free radicals and metabolism associ-
Genome sequence of the radioresistant bacterium Deinococ- ated with the acquisition and loss of desiccation tolerance in
cus radiodurans R1. Science, 286, 1571–1577 (1999). developing seeds. Biochem. Soc. Trans., 24, 451–455 (1996).
21. Claiborne, A. and Fridovich, I.: Purification of the o-di- 37. He, Y. Y. and Hader, D.: Reactive oxygen species and UV-B:
ansidine peroxidase from Escherichia coli B. Physiochemical effect on cyanobacteria. Photochem. Photobiol. Sci., 1, 729–
characterization and analysis of its dual catalatic and peroxi- 736 (2002).You can also read
