Rapid Communication J. Biochem. 134, 25-29 (2003) D O I: 10.1093/j b/mvg l09

Alkyl Hydroperoxide Reductase Dependent on Thioredoxin-Like Protein from Pyrococcus horikoshii

Yasuhiro Kashima and Kazuhiko Ishikawa* Special Division of Human Life Technology, National Institute of Advanced Industrial Science and Technology (AIST Kansai)1-8-31 Midorigaoka, Ikeda, Osaka 563-8577

Received February 25, 2003; accepted May 14, 2003

Pyrococcus horikoshii is an obligate anaerobic hyperthermophilic archaeon. In P. horikoshii cells, a hydroperoxide reductase homologue ORF (PH1217) was found to be induced by oxygen. The recombinant protein, which was expressed in E. coli under aerobic conditions, exhibited no activity. However, the recombinant protein prepared under semi-anaerobic conditions exhibited alkyl hydroperoxide reductase activity. Furthermore, it was clarified that it was coupled with the thioredoxin-like system in P. horikoshii. Western blot analysis revealed that the protein was induced by oxygen and . This protein seems to be sensitive to oxygen but forms a thioredoxin-dependent system to eliminate reactive oxygen species in P. horikoshii.

Key words: alkyl hydroperoxide reductase, hyperthermophilic archaea, oxidative stress, Pyrococcus horikoshii, thioredoxin system.

Organisms have developed various mechanisms that AhpC-like plays a critical role in the elimination have the ability to eliminate many forms of physiological of hydrogen peroxide that is produced through reduction and chemical stress from their environments, such as of the anion by SOR. However, an enzyme for reactive oxygen species (ROS), temperature, pH, and the elimination of peroxide has not been reported in osmotic pressure. In particular, oxidative stress caused hyperthermophilic archaea. In this paper, we report that by ROS has to be eliminated in all living creatures on a protein is induced by oxygen and may be involved in the earth, on which oxygen exists abundantly. Although, the peroxide detoxification system in P horikoshii cells. elimination ability varies between aerobic and anaerobic We investigated the response of intracellular proteins organisms, a mechanism for such elimination is essential in P horikoshii cells to oxygen-stimulation to clarify the for all organisms, because ROS, such as the superoxide resistance systems against ROS. P. horikoshii cells were anion (00, hydrogen peroxide (H2O2), and alkyl cultured in medium containing, 25 g of polypeptone, 5 g hydroperoxide, are produced in cells if there is oxygen in of yeast extract, 25 g of NaCl, 0.75 g of CaC12, 1mg of the environment. ROS are cellular oxidizing substances resazurin, 5mg of citric acid, 100ml of solution A [35g of that can injure DNA, proteins, and membrane lipids. MgSO4•E7112O, 27.5 g ofmgCl2•E6H2O, 3.2g of KCl, 0.5 g of Therefore, organisms have mechanisms involving some NaBr, 0.15 g of H3BO3, 75mg of SrCl2•E6H2O, 1mg of KI, 5 proteins such as (SOD) (1-3), g of KH2PO4 and 20mg of (NH4)2Ni(SO4)2•E6H2O/liter], superoxide reductase (SOR) (4), catalase (5), peroxire and 10ml of solution B [1.5g of nitrilotriacetic acid, 3g of doxin (6), thioredoxin and glutathione-systems (7-12), MgSO4•E7H2O, 0.5g of MnSO4•ExH2O, 1 g of NaCl, 0.1 g of and damage-repairing to eliminate ROS (13-14) FeSO4•E7H2O, 0.1 g of CoSO4•E7H2O, 0.1 g of CaC12•E2H2O, for the protection of their cells. Alkyl hydroperoxide 0.1 g of ZnS04•E7H2O, 10mg of CuSO4•E5H2O, 10mg of reductase (AhpC) has been reported in aerobic and AIK(SO4)2, 10mg of H3BO3, 10mg of Na2MOO4•E2H2O1 10 anaerobic bacteria such as Salmonella typhimurium (15), mg of Na2WO4•E2H2O, and 2mg of Na2SeO3/liter] per liter,. Clostridium pasteurianum (16), Mycobacterium tubercu The pH was adjusted to a final 6.8. The medium was sat losis (17), Hericobacter pylori (18), and E. coli (19), in con urated with nitrogen and then sterilized by autoclaving. jugation with alkyl hydroperoxide reductase subunit F Sterile sulfur (3.0 g/liter) and 2.5 ml of a 20% Na2S solu (AhpF) (20-22) or thioredoxin-like systems (16, 18). This tion were added to the medium after cooling to 90•Ž. The enzyme eliminates damaging substances, such as hydro culture was grown in a 1-liter bottle at 95•Ž under anaer gen peroxide and alkyl hydroperoxide. Not only for aer obic conditions. Cell growth was monitored as the turbid obes but also for anaerobic microorganisms, in particular, ity at 600 nm. At the early logarithmic phase (9 h), 20 ml the detoxification of peroxide is necessary for the systems of air was injected into the culture bottles, followed by that eliminate ROS. shaking. A further 60 min incubation at 95•Ž was per Pyrococcus species are obligate anaerobic and hyper formed, and then the cells were harvested by centrifuga thermophilic archaea. The detoxification mechanisms for tion after cooling down to 4•Ž. The cell paste was resus ROS have been reported to be SOR-rubredoxin systems pended in a 2.5% NaCl solution, and then re-centrifuged. in P furiosus (4, 23-24). It has been expected that an The pellet was homogenized in 20mM Tris-HCl (pH 8.0) by ultrasonication, and then centrifuged. The proteins in the supernatant were precipitated by ammonium sulfate *To whom correspondence should be addressed. Tel: +81-727-51 precipitation (90% saturation), and then dialyzed against 9526, Fax: +81-727-51-9628, E-mail: [email protected] 5mM Tris-HCl (pH 8.0). The proteins (0.5mg) were sep

Vol. 134, No. 1, 2003 25 ? 2003 The Japanese Biochemical Society, 26 Y. Kashima and K. Ishikawa

Fig. 1. Alignment of the amino acid sequences of P. horikoshii AhpC (PH1217 protein) and some bacterial AhpCs. Dashes within sequences represent gaps introduced to optimize alignment. The positions of the N-terminal Cys residue conserved in all AhpCs are boxed, and those of the C-ter minal Cys residues are given in bold. Dots and asterisks under the sequence indicate the same amino acid in the three or four proteins, respectively.

arated by isoelectric focusing using ReadyStrip IPG E. coli Rosetta(DE3) harboring the constructed plas strips (pH 3-11, 11 cm length; Biorad, Hercules, CA, mid was grown in LB broth supplemented with 0.4% glu USA). The second electrophoresis was performed on a cose at 37•Ž under the following two different conditions. 15% SDS-polyacrylamide gel. As a control, a cell homo Under aerobic conditions, cells were grown until O.D.600 genate prepared by the same procedure without air-stim = 0.6 with shaking, and then the recombinant protein ulation was also electrophoresed. After staining with was induced with 1mM isopropyl-1-thio-ƒÀ-D-galactopyra Coomassie-Brilliant Blue R250, an increasing protein noside (IPTG) for 3 h. Under semi-anaerobic conditions, band was only detected when the cells were stimulated cells were grown aerobically until OD600 = 0.3, and then by air (data not shown). The N-terminal amino acid the bottle was capped and sealed. After further incuba sequence of the protein was determined to be VVIGEK, tion for 1 h at 37•Ž without shaking, the recombinant and PH1217 in the genome database of P horikoshii protein was induced with 1mM IPTG for 16 h. The two (http://www.bio.nite.go jp:8080/dogan/MicroTop) was found kinds of cell paste obtained on centrifugation at 7,000 to be a corresponding protein. PH1217 was predicted to be a DNA encoding alkyl hydroperoxide reductase (AhpC) from the sequence homology. The deduced amino acid sequence of the PH1217 protein contains three cysteine residues (Fig. 1). The consensus sequence around Cys46 of the PH1217 protein (DFTPVCTTE) exhibits high homology to the sequence of other AhpCs (DFTFVCPTE). The C-terminal region of the protein is larger than those of the other AhpCs, and the region around the other two cysteine residues (Cys205 and Cys211) exhibits no homology to those of the others. The PH1217 protein induced by oxygen may play an impor tant role in the resistance against oxygen. To characterize the PH1217 protein, we cloned the gene and prepared a recombinant protein using E. coli. The complete coding sequence of PH1217 was amplified by 30 cycles of PCR [denaturing at 96•Ž, annealing at 60•‹ and extension at 74•Ž, with KOD DNA polymerase (Toyobo, Osaka)] using the genomic DNA of P horikoshii as a template. Primers, 5•Œ-GAGGAGATCCATATGGTAG TGATTGGAGAA-3•Œ and 5•Œ-GGAAGGAATTCATTCAAG CTTCTTATAGCAGA-3•Œ, containing recognition sites for restriction enzymes (underlined) were used for amplifica tion of the gene. The resulting DNA fragment was digested with NdeI and EcoRI (Takara, Kyoto), and then inserted into the corresponding site of expression vector pET21a (Novagen, Madison, WI, USA). The plasmid obtained was introduced into E. coli Rosetta(DE3) (Nova gen, Madison, WI, USA). The nucleotide sequence of the inserted gene was confirmed by DNA sequencing. Prepa ration of the genomic DNA of P horikoshii was reported previously (25).

J. Biochem . AhpC like protein from P horikoshii 27

rpm for 5 min were re-suspended in 20mM Tris-HCl (pH8.0), and then homogenized by ultrasonication. The homogenates were treated at 85•Ž for 30 min, and then centrifuged at 15,000 rpm for 20 min. The supernatants were dialyzed against 20mM Tris-HCl (pH8.0). All extraction steps were performed under nitrogen gas, and de-gassed and N2-saturated buffer was used. From the E. coli cells under both conditions, the recombinant PH1217 Fig. 3. Proposed pathway for the transfer of reducing equiv protein, of which the molecular weight was 25 kDa, was alents from NADPH to hydroperoxide in P. horikoshii. prepared (Fig. 2A). We recently found two redox proteins that form a thioredoxin-like system composed of homo logues of (PhRP) and thioredoxin reductase reductase activity in the obtained crude enzyme solutions (PhTrxR) in P. horikoshii (manuscript submitted). The was examined (Fig. 2B). When the PH1217 protein was recombinant PhRP and PhTrxR were expressed with the expressed in E. coli cells under aerobic conditions, no degradation of cumene hydroperoxide was observed (Fig. pET vector system in E. coli. The alkyl hydroperoxide 2B). This means that the expressed protein exhibited no alkyl hydroperoxide reductase activity. On the other Fig. 2. SDS-PAGE profile of the enzyme solutions used for hand, the protein prepared under semi-anaerobic condi measurement of the enzyme activity (A), alkyl hydroperox tions exhibited DTT-dependent alkyl hydroperoxide ide reductase activity of the protein (B), and the activity in reductase activity. The protein also exhibited NADPH the presence of various components (C). (A) Crude extracts of E. coli harboring aerobically and semi-anaerobically expressed - dependent alkyl hydroperoxide reductase activity in the PH1217 protein in E. coli were extracted and treated at 85•Ž or 30 presence of PhRP and PhTrxR (Fig. 2, B and C). In addi min. After centrifugation (15,000 rpm, 20 min), the supernatants tion, there is no AhpF homologous protein in the P (2.5 ƒÊg of protein) were loaded onto a 17% polyacrylamide gel. M, horikoshii genome. These results indicated that the molecular weight markers: phosphorylase b (97 kDa), albumin from PH1217 protein (hereafter referred to as PhAhpC) pre bovine serum (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa), and ƒ¿-lactalbumin (14.4 kDa); 1, pared under semi-anaerobic conditions exhibited thiore Extract of aerobic cultured cells; 2, Extract of semi-anaerobic cul doxin-like protein-dependent peroxidase activity (Fig. 3), tured cells. (B) For measurement of DTT-dependent activity, the as reported for Helicobacter pyloli (18). Basically, P reaction mixture contained 50mM MOPS (pH 7.0), 20 ƒÊM aerobi horikoshii does not seem to have strong resistance sys cally (open squares) or semi-anaeeeeeobically (solid squares) produced tems for high concentrations of oxygen such as that in air, PH1217 protein, 0.3mM DTT, and 1mM cumene hydroperoxide. because of its obligate anaerobic characteristics. Accord The reaction mixture for PhRP-dependent activity contained 50 ingly, PhAhpC seems to be inactivated by air, probably mM MOPS (pH 7.0), 20 ƒÊM aerobically (open circles) or semi-anaer obically (solid circles) produced PH1217 protein, 10 ƒÊM PhRP, 50 oxygen. nM PhTrxR, 0.3mM NADPH, and 1mM cumene hydroperoxide. As a negative control, a mixture contained 50mM MOPS (pH 7.0), and 1mM cumene hydroperoxide was also prepared. The reaction mix tures were incubated at 85•Ž, and aliquots were removed at the indicated times. The results are expressed as the decrease-rate in the reaction subtracted from that in the negative control. To moni tor the disappearance of cumene hydroperoxide from the solution, experiments were conducted essentially as described elsewhere (18). Reactions were terminated by the addition of 0.95 ml of 10% Fig. 4. Western blot analysis of PhAhpC expression. For the

trichloroacetic acid to 50 ƒÊl of the reaction mixture, and then to preparation of anti-PhAhpC antisera, the PhAhpC protein was determine the peroxide content as ferrithiocyanate complex forma purified to homogeneity as mentioned in the legend to Fig. 2. Antis tion, 0.2 ml of 10mM Fe(NH4)2(SO4)2 and 0.1 ml of 2.5 M KSCN era against PhAhpC were obtained using the purified protein in were added. After centrifugation to remove the precipitated protein, Freund's adjuvant. 50ƒÊg of PhAhpC protein was injected into red ferrithiocyanate complex was measured at 480 nm, and the per BALB/c mice. Booster immunizations were given weekly under the oxide concentration was determined from a standard curve same conditions but with the use of incomplete Freund's adjuvant obtained with cumene hydroperoxide. The activity was expressed as until anti-PhAhpC antibodies were detected in the sera. It was con the decrease in cumene hydroperoxide in the reaction mixtures. The firmed that the obtained antisera did not bind to protein in the results are the averages of four trials. The values are the means of extract of E. coli Rosetta(DE3) on ELISA. The cells of P horikoshii four experiments •} SD (C) Each component was present (+) or were stimulated with 20 ml of air at the early logarithmic phase (9 absent (-). Conditions: 1mM cumene hydroperoxide, 0.3mM h) for 0 (lane A), 30 (with air; lane B), and 60 min (with air, lane C), NADPH, 20 ƒÊM PhAhpC, 10 ƒÊM PhRP, and 50nM PhTrxR were and with 1mM hydrogen peroxide for 30 min (lane D). The cells incubated in 50mM MOPS (pH 7.0) at 85•Ž, and then the concen were harvested and homogenized in 20mM Tris-HCl (pH 8.0). In tration of cumene hydroperoxide was determined as ferrithiocy the case of stimulation with hydrogen peroxide, the cells were

anate complex formation. The concentration of the anaerobically grown in the medium without sulfur. Extracts of the cells were ana expressed PhAhpC was determined by determination of the density lyzed by Western blotting. Following SDS-PAGE of the homoge of the band on SDS-PAGE with reference to that of the aerobically nates, proteins were electrophoretically transferred to polyvinyli expressed PhAhpC protein purified to homogeneity by salting out, dene difluoride (PVDF) membranes. The membranes were blocked Hi-Trap Q (Amersham Biosciences, Picataway, NJ, USA), and Hi for 1 h with a blocking solution (Roche Diagnostics, Basel, Switzer load phenyl (Amersham biosciences, Picataway, NJ, USA). Compar land). The membranes were then incubated with the mouse anti- ison of the densities of two bands was performed with an Gel-Pro PhAhpC antisera in PBS, followed by incubation with alkaline

analyzer (MediaCybernetics, Silver Spring MD, USA). The results phosphatase-conjugated goat anti-mouse IgG (Sigma, St. Louis, are presented as relative activity as to the reaction with all the com MO, USA). The antibody to PhAhpC was detected using an AP con

ponents, and are the averages of four trials. 100% activity corre jugate kit (Biorad, Hercules, CA, USA). Cell extracts (9 ƒÊg sponds to a decrease of 0.12mM cumene hydroperoxide per 30 min. each) were loaded on a 15% gel.

Vol. 134, No. 1, 2003 28 Y. Kashima and K. Ishikawa

We investigated how the expression of PhAhpC was 8. Williams, C.H., Arscott, L.D., Muller, S., Lennon, B.W., Lud affected by oxygen (Fig. 4, lanes B and C). PhAhpC was wig, M.L., Wang, P.E, Veine, D.M., Becker, K., and Schirmer, R.H. (2000) Thioredoxin reductase. Two modes of apparently increased in P horikoshii cells stimulated by have evolved. Eur. J Biochem. 267, 6110-6117 air. In addition, PhAhpC was induced by hydrogen perox 9. Holmgren, A. (1979) Reduction of disulfide by thioredoxin. J. ide (Fig. 4, lane D) like Prx from yeast (26). However, the Biol. Chem. 254, 9113-9119 induction of the PhAhpC was slight because most of the 10. Song, J.J., Rhee, J.G., Suntharalingam, M., Walsh, S.A., Spitz, hydrogen peroxide might have reacted with the sodium D.R,, and Lee, Y.J. (2002) Role of glutaredoxin in metabolic oxi sulfate that had been added to the medium to eliminate dative stress. Glutaredoxin as a sensor of oxidative stress mediated by H2O2. J Biol. Chem. 277,46566-46575 oxygen. In the genome database of P horikoshii, catalase 11. Holmgren, A. and Aslund, F. (1995) Glutaredoxin. Methods related proteins were not found. These results suggest Enzymol. 252,283-292 that the expression of PhAhpC is also induced by hydro 12. Pueyo, C., Jurado, J., Prieto-Alamo, M.J., Monje-Casas, F., and gen peroxide produced through reduction of the superox Lopez-Barea, J. (2002) Multiplex reverse transcription ide anion via SOR. polymerase chain reaction for determining transcriptional reg In P. furiosus, an SOR-rubredoxin system for elimina ulation of thioredoxin and glutaredoxin pathways. Methods tion of the oxygen anion has been reported (23-24). P Enzymol. 347,441-451 horikoshii also has a homologue of SOR (PH1083; 74% 13. Imalay, J. and Linn, S. (1988) Toxic DNA damage by hydrogen peroxide through the Fenton reaction in vivo and in vitro. Sci identity to SOR of P furiosus). Antioxidant systems may ence 240, 640-642 be critical for the defense against ROS generated by oxy 14. Jamieson, D. and Storz, G. (1997) Transcriptional regulators of gen also for P. horikoshii. Therefore, the Trx system oxidative stress responses in Oxidative Stress and Molecular dependent AhpC is also important for the antioxidant Biology of Antioxidant Defenses. (Scandalios. J., ed.) pp. 91 system in Pyrococcus species. Interestingly, no homo -115, Cold spring harbor Laboratory, Cold Spring Harbor, NY logues of catalase or glutathione reductase systems such 15. Jacobson, F.S., Morgan, R.W., Christman, M.F., and Ames, B.N. (1989) An alkyl hydroperoxide reductase from Salmonella as gamma-L-glutamyl-L-cysteine synthethase and glu typhimurium involved in the defense of DNA against oxidative tathione reductase were found in the genome of Pyrococ damage. J Biol. Chem. 264,1488-1496 cus species. Therefore, P horikoshii does not have other 16. Reynolds, C.M., Meyer, J., and Poole, L.B. (2002) An NADH main reductase systems such as glutathione-dependent - dependent bacterial thioredoxin reductase-like protein in con redox system (11-13). Accordingly, PhAhpC presumably junction with a glutaredoxin homologue form a unique perox iredoxin (AhpC) reducing system in Clostridium pasteur plays an important role in the resistance against oxygen, ianum. Biochemistry 41, 1990-2001 even if it is inactivated by oxygen. Furthermore, anaero 17. Hillas, P.J., del Alba, F.S., Oyarzabal, J., Wilks, A., and de Mon bic conditions for the production of recombinant proteins tellano, P.R.O. (0000) The AhpC and AhpD antioxidant defense from Pyrococcus species should be important for clarify system of Mycobacterium tuberculosis. J Biol. Chem. 275, ing the mechanism of resistance against oxygen, as 18801-18809 described here. 18. Baker, L.M.S., Raudonikiene, A., Hoffman, P.S., and Poole, L.B. 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