The AhpC and AhpD Antioxidant Defense System of Mycobacterium tuberculosis
2000; Elsevier BV; Volume: 275; Issue: 25 Linguagem: Inglês
10.1074/jbc.m001001200
ISSN1083-351X
AutoresPatrick J. Hillas, Federico Soto del Alba, Julen Oyarzábal, Angela Wilks, Paul R. Ortiz de Montellano,
Tópico(s)Trace Elements in Health
ResumoThe peroxiredoxin AhpC from Mycobacterium tuberculosis has been expressed, purified, and characterized. It differs from other well characterized AhpC proteins in that it has three rather than one or two cysteine residues. Mutagenesis studies show that all three cysteine residues are important for catalytic activity. Analysis of the M. tuberculosis genome identified a second protein, AhpD, which has no sequence identity with AhpC but is under the control of the same promoter. This protein has also been cloned, expressed, purified, and characterized. AhpD, which has only been identified in the genomes of mycobacteria and Streptomyces viridosporus, is shown here to also be an alkylhydroperoxidase. The endogenous electron donor for catalytic turnover of the two proteins is not known, but both can be turned over with AhpF fromSalmonella typhimurium or, particularly in the case of AhpC, with dithiothreitol. AhpC and AhpD reduce alkylhydroperoxides more effectively than H2O2 but do not appear to interact with each other. These two proteins appear to be critical elements of the antioxidant defense system of M. tuberculosis and may be suitable targets for the development of novel anti-tuberculosis strategies. The peroxiredoxin AhpC from Mycobacterium tuberculosis has been expressed, purified, and characterized. It differs from other well characterized AhpC proteins in that it has three rather than one or two cysteine residues. Mutagenesis studies show that all three cysteine residues are important for catalytic activity. Analysis of the M. tuberculosis genome identified a second protein, AhpD, which has no sequence identity with AhpC but is under the control of the same promoter. This protein has also been cloned, expressed, purified, and characterized. AhpD, which has only been identified in the genomes of mycobacteria and Streptomyces viridosporus, is shown here to also be an alkylhydroperoxidase. The endogenous electron donor for catalytic turnover of the two proteins is not known, but both can be turned over with AhpF fromSalmonella typhimurium or, particularly in the case of AhpC, with dithiothreitol. AhpC and AhpD reduce alkylhydroperoxides more effectively than H2O2 but do not appear to interact with each other. These two proteins appear to be critical elements of the antioxidant defense system of M. tuberculosis and may be suitable targets for the development of novel anti-tuberculosis strategies. polymerase chain reaction dithiothreitol polyethyleneimine potassium phosphate buffer high pressure liquid chromatography Tuberculosis, caused by opportunistic infection byMycobacterium tuberculosis, is a leading cause of death (1.Evans J. Chemistry in Britain. 1998; 34: 38-42Google Scholar). Worldwide, infection rates are increasing although in the United States the rate of tuberculosis infection has begun to decrease after an increase in the late 1980s (2.Snider D.E.J. Roper W.L. N. Engl. J. Med. 1992; 326: 703-705Crossref PubMed Scopus (373) Google Scholar). A very alarming observation is the appearance of M. tuberculosis strains resistant to many of the front-line compounds, including isoniazid, that are currently utilized to treat the disease (3.Zumla A. Grange J. Br. Med. J. 1998; 316: 1962-1964Crossref PubMed Scopus (38) Google Scholar). Middlebrook and co-workers (4.Middlebrook G. Cohn M.C. Shaffer W.B. Am. Rev. Tuberc. Pulm. Dis. 1954; 70: 852-872Crossref PubMed Google Scholar) observed in the 1950s that M. tuberculosis strains resistant to isoniazid were devoid of catalase/peroxidase activity. This circumstantial link between peroxidase and isoniazid activities was placed on a molecular footing by Heym et al. (5.Heym B. Zhang Y. Poulet S. Young D. Cole S.T. J. Bacteriol. 1993; 175: 4255-4259Crossref PubMed Scopus (192) Google Scholar, 6.Heym B. Alzari P.M. Honore N. Cole S.T. Mol. Microbiol. 1995; 15: 235-245Crossref PubMed Scopus (313) Google Scholar), who showed that isoniazid-resistant M. tuberculosis strains had deletions or mutations in the katG gene that encodes for the KatG catalase/peroxidase. The importance of KatG in the action of isoniazid was confirmed by the demonstration that transformation ofEscherichia coli or Mycobacterium smegmatis, both of which are isoniazid resistant, with the katG gene rendered them sensitive to isoniazid (7.Zhang Y. Garbe T. Young D. Mol. Microbiol. 1993; 8: 521-524Crossref PubMed Scopus (157) Google Scholar). Furthermore, Johnson and Schultz (8.Johnson K. Schultz P.G. J. Am. Chem. Soc. 1995; 116: 7425-7426Crossref Scopus (271) Google Scholar) showed that isoniazid is oxidized by the M. tuberculosis KatG catalase/peroxidase to a number of chemically reactive products. These combined results imply that isoniazid is a prodrug that must be processed into its active form by the bacterial cell. The critical target of activated isoniazid is not yet clear. Evidence exists that this role is played by the inhA gene product, an enoyl reductase (9.Benerjee A. Dubnau E. Quernard A. Balasubramanian V. Um K.S. Wilson T. Collins D. de Lisle G. Jacobs Jr., W.R. Science. 1994; 263: 227-230Crossref PubMed Scopus (1236) Google Scholar), and/or by KasA, a β-ketoacyl synthase (10.Mdluli K. Slayden R.A. Zhu Y. Ramaswamy S. Pan X. Mead D. Crane D.D. Musser J.M. Barry III, C.E. Science. 1998; 280: 1607-1610Crossref PubMed Scopus (375) Google Scholar). Both of these enzymes are involved in the biosynthesis of mycolic acid, an essential constituent of the M. tuberculosis cell wall. It is not yet clear whether one or both of these proteins is the principal target, or whether there are additional targets for activated isoniazid (11.Mdluli K. Sherman D.R. Hickey M.J. Kreiswirth B.N. Morris S. Stover C.K. Barry C.E. J. Infect. Dis. 1996; 174: 10895-10900Crossref Scopus (102) Google Scholar, 12.Barry C.E.I. Slayden R.A. Mdluli K. Drug Resistance Updates. 1998; 1: 128-134Crossref PubMed Scopus (20) Google Scholar). In view of the requirement for the activation of isoniazid by the KatG catalase/peroxidase, one strategy used by the organism to overcome its sensitivity to isoniazid is to suppress the catalase/peroxidase activity through mutation of the katG gene. However, the survival of the bacterium requires that it compensate in some manner for loss of the catalase/peroxidase, as it must still attenuate the oxidative stress caused by peroxides and other reactive oxygen species.M. tuberculosis primarily resides in the macrophages of the host, where it is subjected to a highly oxidative environment (13.Fenton M.J. Vermeulen M.W. Infection and Immunology. 1996; 64: 683-690Crossref PubMed Google Scholar, 14.Chan J. Kaufmann S.H.E. Bloom B.R. Immune Mechanisms of Protection. Tuberculosis: Pathogenesis, Protection, and Control. ASM Press, Washington, D. C.1994Google Scholar). This environment includes peroxides formed by the oxidative burst, species such as peroxynitrite formed by the inducible nitric oxide synthase, and the alkyl peroxides that result from exposure of unsaturated lipids to oxidative stress. Analysis of the genes induced in isoniazid-resistant M. tuberculosis indicates that one of the mechanisms used by the organism to compensate for loss of the KatG antioxidant activity is to up-regulate the ahpC gene product, which codes for a non-hemoprotein alkylhydroperoxidase (15.Sherman D.R. Mdluli K. Hickey M.J. Arain T.M. Morris S.L. Barry C.E. Stover C.K. Science. 1996; 272: 1641-1643Crossref PubMed Scopus (374) Google Scholar, 16.Sherman D.R. Sabo P.J. Hickey M.J. Arain T.M. Mahairas G.G. Yuan Y. Barry C.E. Stover C.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6625-6629Crossref PubMed Scopus (178) Google Scholar, 17.Chae H.Z. Robison K. Poole L.B. Church G. Storz G. Rhee S.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7017-7021Crossref PubMed Scopus (706) Google Scholar, 18.Deretic V. Pagan-Ramos E. Zhang Y. Dhandayuthapani S. Via L.E. Nature Biotechnol. 1996; 14: 1557-1561Crossref PubMed Scopus (52) Google Scholar, 19.Zhang Y. Dhandayuthapani S. Deretic V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13212-13216Crossref PubMed Scopus (100) Google Scholar). Incubation of M. tuberculosis expressing elevated levels of the alkylhydroperoxidase with isoniazid has shown that the drug is not activated by this enzyme (15.Sherman D.R. Mdluli K. Hickey M.J. Arain T.M. Morris S.L. Barry C.E. Stover C.K. Science. 1996; 272: 1641-1643Crossref PubMed Scopus (374) Google Scholar). AhpC thus differs from KatG in its interactions with isoniazid. Very little is known about the M. tuberculosisalkylhydroperoxidase in terms of its structure or catalytic mechanism, as the protein has not yet been purified and investigated. The most studied member of the alkylhydroperoxidase family is the enzyme fromSalmonella typhimurium (20.Ellis H.R. Poole L.B. Biochemistry. 1997; 36: 13349-13356Crossref PubMed Scopus (177) Google Scholar, 21.Ellis H.R. Poole L.B. Biochemistry. 1997; 36: 13357-13364Crossref PubMed Scopus (47) Google Scholar). This protein contains two cysteine sulfhydryls that catalyze the reduction of peroxides to the corresponding alcohols and water with concomitant oxidation of the cysteine residues to give a disulfide bond (SchemeFS1). A sulfenic acid derivative of one of the two sulfhydryl groups is thought to be a transient intermediate in the formation of the disulfide link (22.Yeh J.E. Claiborne A. Hol W.G.J. Biochemistry. 1996; 35: 9951-9957Crossref PubMed Scopus (89) Google Scholar, 23.Ellis H.R. Poole L.B. Biochemistry. 1997; 36: 15013-15018Crossref PubMed Scopus (210) Google Scholar). The catalytic cycle is completed by reduction of the disulfide bond using AhpF, a flavoprotein reductase (Scheme FS2) (21.Ellis H.R. Poole L.B. Biochemistry. 1997; 36: 13357-13364Crossref PubMed Scopus (47) Google Scholar). The protein required to reduce the AhpC disulfide bond is not the same in all organisms. Thus, in yeast the alkylhydroperoxidase is coupled to thioredoxin and thioredoxin reductase rather than to an AhpF-like protein (24.Chae H.Z. Chung S.J. Rhee S.G. J. Biol. Chem. 1994; 269: 27670-27678Abstract Full Text PDF PubMed Google Scholar).Figure FS2View Large Image Figure ViewerDownload Hi-res image Download (PPT) The M. tuberculosis genome has been sequenced in its entirety (25.Hamlin N. Holroyd S. Hornsby T. Jagels K. Krogh A. McLean J. Moule S. Murphy L. Oliver K. Osborne J. Quail M.A. Rajandream M.-A. Rogers J. Rutter S. Seeger K. Skelton J. Squares S. Squares R. Sulston J.E. Taylor K. Whitehead S. Barrell B.G. Nature. 1998; 393: 537-544Crossref PubMed Scopus (6557) Google Scholar). No ahpF gene appears to be present, although a number of flavoproteins of unknown function are found in the genome. In S. typhimurium the ahpF gene is found immediately downstream of the ahpC gene. The corresponding position in the M. tuberculosis genome is occupied by a gene that, because of its position in the sequence, was named ahpD. AhpD exhibits no sequence similarity with either ahpCor ahpF and its function is unknown. We report here the first expression and purification of the M. tuberculosisAhpC and AhpD proteins and their initial structural and catalytic characterization. Oligonucleotide synthesis and DNA sequencing were performed by the Biomolecular Resource Center of the University of California, San Francisco. A Perkin-Elmer 480 DNA thermal cycler was used for PCR1 experiments. The pGem-T and pET23a plasmids were from Novagen (Madison, WI). Plasmid pACYC was from New England Biolabs (Beverly, MA). Restriction enzymes and Vent DNA polymerase were purchased from New England Biolabs and Promega (Madison, WI). Plasmids were purified using the Qiagen (Chatsworth, CA) Quick-Prep kit. E. coli strain BL21(DE3) was from Novagen and strain DH5α from Life Technologies, Inc. (Gaithersburg, MD). Q-Sepharose Fast Flow was from Amersham Pharmacia Biotech and the Ni-NTA-agarose resin was from Qiagen. PEI was from Research Biotechnologies, Inc. (Natick, MA). LB medium was from Life Technologies, Inc. All other chemical reagents were purchased from Sigma. An Amersham Pharmacia Biotech Sephadex 200 column, connected to an Amersham Pharmacia Biotech PCC-500 FPLC system, was used to determine the native aggregation state of each protein. A Hewlett-Packard HP-8452 UV-visible spectrophotometer was used for all spectroscopic measurements. The plasmid encoding for the S. typhimurium AhpF (pAF1) was generously provided by Leslie B. Poole (26.Poole L.B. Biochemistry. 1996; 35: 65-75Crossref PubMed Scopus (108) Google Scholar). The plasmids encoding the M. tuberculosis thioredoxin and thioredoxin reductase were a gift from Brigitte Wieles (27.Wieles B. Nagai S. Wiker H.G. Harboe M. Ottenhoff T.H.M. Infect. Immun. 1995; 63: 4946-4948Crossref PubMed Google Scholar, 28.Wieles B. van Noort J. Drijfhout J.W. Offringa R. Holmgren A. Ottenhoff T.H.M. J. Biol. Chem. 1995; 270: 25604-25606Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). All expression plasmids were introduced into competent BL21(DE3)E. coli. The ahpC gene was generously provided by Clifton E. Barry as a 1.3-kilobase NotI-PstI fragment in pMH91 (16.Sherman D.R. Sabo P.J. Hickey M.J. Arain T.M. Mahairas G.G. Yuan Y. Barry C.E. Stover C.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6625-6629Crossref PubMed Scopus (178) Google Scholar). The open reading frame for the ahpC gene was amplified by PCR with the following primers (forward: 5′-CGCTAGGTACCATATGCCACTGCTAACCATTGGC-3′; reverse: 5′-TCTAGAGGATCCTTAGGCCGAAGCCTTGAGGAG-3′). The primers coded for an NdeI restriction site at the ATG codon and aBamHI site following the stop (TAA) codon. The reaction contained 50 ng of pMH91, 50 pmol each of the primers, 1 mmdNTPs, and 10 units of Vent DNA polymerase (NEB) in a final volume of 50 μl of 20 mm Tris-HCl (pH 8.8), 10 mm KCl, 10 mm (NH4)2SO4, 2 mm MgSO4, and 0.1% Triton X-100. The annealing and extension cycles were as follows: 94 °C for 10 min (1 cycle), 94 °C for 1 min, 50 °C for 1 min, 72 °C for 1 min (10 cycles), 94 °C for 1 min, 65 °C for 1 min, 72 °C for 1 min (20 cycles), and 72 °C for 10 min (1 cycle). Following gel purification of the amplified product the 0.59-kilobase gene was digested withNdeI and BamHI, ligated into pGem-T, and subcloned into pET 23a. This plasmid was called pEahpC. A second vector was constructed in which a 6-His tag was added to the 3′ end of the ahpC gene using the primer 5′-TCTAGACTCGAGGGCCGAAGCCTTGAGGAG-3′. This primer encoded for anXhoI site that removed the stop codon. The PCR conditions were identical to the above reaction. This plasmid was called pEahpC-histag. The open reading frame for the ahpD gene was amplified by PCR from M. tuberculosis genomic DNA (provided by Clifton E. Barry) using the following primers (forward: 5′-GATCTGGTTGCCCGGGAACATATGAGTATAGAAAAGCTC-3′; reverse: 5′-GGCGTCATGGCGTCGACACACTTAGCTTGGGCTTAGTGCCTCGGTTGTGCC-3′). The primers coded for an NdeI restriction site at the ATG codon and a SalI site following the stop (TAA) codon. The reaction contained 50 ng of M. tuberculosis genomic DNA, 50 pmol each of the primers, 2 mm dNTPs, and 10 units of Vent DNA polymerase in a final volume of 100 μl of 20 mmTris-HCl (pH 8.8), 10 mm KCl, 10 mm(NH4)2SO4, 2% dimethyl sulfoxide, and 0.1% Triton X-100. The annealing and extension cycles were as follows: 90 °C for 10 min (1 cycle), 90 °C for 1 min, 72 °C for 1 min, 60 °C for 1 min (30 cycles), and 72 °C for 10 min (1 cycle). Following gel purification of the amplified product the 0.55-kilobase gene was digested with NdeI andSalI. The ahpD gene was then inserted into pACYC using the available NdeI and SalI sites. This plasmid was called pACahpD. The single cysteine to serine mutants of the 3 cysteine residues of AhpC and 2 cysteine residues of AhpD were made using the QuikChange Site-directed Mutagenesis Kit from Stratagene (La Jolla, CA). The PCR conditions were as follows: 50 ng of pEahpC (without his-tag) or pACahpD, 125 ng of each primer, 50 μm each dNTP, 2.5 units of Pfu polymerase, and 5 μl of 10× Pfu buffer in a total volume of 50 μl. The cycling parameters were: 95 °C for 30 s (1 cycle), 95 °C for 30 s, 55 °C for 1 min, and 68 °C for 8.4 min (16 cycles). After amplification the PCR mixture was incubated with 20 units of DpnI for 1 h at 37 °C and then 1 μl was used to transform 50 μl of DH5α cells. The mutations where confirmed by DNA sequencing. The primers used for the mutagenesis were: C61S, forward primer: 5′-TTCACGTTCGTGTCCCCTACCGAG-3′, reverse primer: 5′-CTCGGTAGGGGACACGAACGTGAA-3′; C174S, forward primer: 5′-GACGAGCTGTCCGCATGCAACTGG-3′, reverse primer: 5′-CCAGTTGCATGCGGACAGCACGTC-3′; C176S, forward primer: 5′-GAGCTGTGCGCATCCAACTGGCGC-3′, reverse primer: 5′-GCGCCAGTTGGATGCGCACAGCAC-3′. In the case of ahpD mutagenesis, the C129S primers were: forward: 5′-GCGATCAACGGGTCCTCGCATTGCCTC-3′, reverse: 5′-GAGGCAATGCGAGGACCC-GTTGATCGC-3′. The primers for the C132S mutations were: forward: 5′-GGGTGCTCGCATTCCCTCGTCGCCCAC-3′, reverse: 5′-GTGGGCGACGAGGGAATGCGAGCACCC-3′. The underlined codons represent the cysteine mutation, with the boldface letters indicating the nucleotide changed to facilitate the mutation. Bacterial growth was carried out at 37 °C in LB medium containing 100 μg/ml ampicillin (for pEahpC) or 50 μg/ml chloramphenicol (for pACahpD and pAF1). One colony was used to inoculate 50 ml of LB medium containing the appropriate antibiotic, and the culture was incubated for 10 h. The culture was used to inoculate a 1-liter culture of LB containing the appropriate antibiotic at a ratio of 10 ml/liter. When the A 600 value of the culture reached 0.7–1.0, isopropyl-β-d-thiogalactopyranoside was added to a final concentration of 0.5 mm for pEahpC and pAF1, and 0.2 mm for pACahpD. Incubation was continued for 3–3.5 h at 37 °C for pEahpC and pAF1, and 20 °C for pACahpD. Cells were harvested by centrifugation at 5000 × g for 45 min, 4 °C, and stored at −20 °C overnight. Cells were suspended in a 4-fold excess (with respect to the initial weight of cells) of lysis buffer (50 mmKPi, pH 7.0, 1.0 mm DTT, 1.0 mmEDTA, 44 μg/ml phenylmethanesulfonyl fluoride, 2 μg/ml leupeptin, 2 μg/ml pepstatin, 5% glycerol, and 5% lysozyme). The solution was stirred 60 min at 4 °C. The cells were then sonicated using a Branson sonicator with 4 bursts of 30 s at 45 W with 30 s intervals. The cell debris was precipitated by centrifugation at 27,000 × g for 60 min at 4 °C. The supernatant was removed and PEI was added to a final concentration of 0.005%. The solution was stirred for 15 min at 4 °C and then centrifuged at 27,000 × g for 15 min at 4 °C. The PEI supernatant was then loaded onto the Q-Sepharose Fast Flow column (1.5 × 12 cm) equilibrated in 50 mm KPi, pH 7.0, 1.0 mm DTT, 1.0 mm EDTA, and 5% glycerol. After loading, the resin was washed with the same buffer for 10 column volumes, followed by a wash with buffer containing 0.2 mKCl for 10 column volumes. The protein was eluted with a gradient from 0.2 to 0.4 m KCl in 50 mm KPi, pH 7.0, 1.0 mm DTT, 1.0 mm EDTA, 5% glycerol. The protein eluted at approximately 0.25 m KCl. Fractions containing pure AhpC, as assessed by denaturing 20% polyacrylamide gels, were pooled, concentrated in an Amicon ultrafiltration cell using a YM10 membrane, and dialyzed against 20 mmKPi, pH 7.0, 50 mm KCl, 0.1 mmEDTA, and 5% glycerol (3 × 2 liter). The protein was stored at −70 °C until used. Cells were suspended in a 6-fold excess (with respect to the initial weight of cells) of lysis buffer. The solution was stirred 60 min at 4 °C. A PEI supernatant was prepared and loaded onto the Q-Sepharose column as described for AhpC. After loading, the resin was washed with the same buffer for 20 column volumes. The protein was eluted with a gradient from 0 to 0.1 m KCl in 50 mm KPi, pH 7.0, 1.0 mm DTT, 1.0 mm EDTA, 5% glycerol. The protein eluted at approximately 0.03 m KCl. Fractions containing pure AhpD, as assessed by denaturing 20% polyacrylamide gels, were pooled, concentrated, and dialyzed against 50 mm KPi, pH 7.0, 100 mm KCl, 0.1 mm EDTA, and 5% glycerol (3 × 2 liter). The protein was stored at −70 °C until used. This enzyme was purified according to the protocol of Poole and Ellis with slight modifications (30.Poole L.B. Ellis H.R. Biochem. 1996; 35: 56-64Crossref PubMed Scopus (182) Google Scholar). Nucleic acids were removed with 0.005% PEI, and the ammonium sulfate precipitation steps were omitted. These enzymes were expressed and purified according to the protocol of Zhang et al. (31.Zhang Z. Hillas P.J. Ortiz de Montellano P.R. Arch. Biochem. Biophys. 1999; 363: 19-26Crossref PubMed Scopus (38) Google Scholar) with a poly-histidine tag on each protein to facilitate purification. Protein size determination was performed using an Amersham Pharmacia Biotech Sephadex 200 FPLC column. The column was equilibrated in 50 mm KPi, pH 7.0, 0.1 mm EDTA, 100 mm KCl, and 5% glycerol at a flow rate of 0.5 ml/min. Approximately 0.5 mg of protein was injected on the column. Protein elution was monitored at a wavelength of 280 nm. Data was collected and processed using the Virtual Bench (National Instruments) software. Rates of hydroperoxide reduction were determined anaerobically in a coupled assay with AhpF, monitoring the decrease in absorbance at 340 nm due to NADH oxidation. The assays typically contained 2 mm hydroperoxide substrate in 100 mmKPi, pH 7.0, 1 mm EDTA, 0.25 mmNADH, and 20 μm either AhpC or AhpD, and 10 μm AhpF. Background NADH oxidation due to AhpF was monitored, then the hydroperoxide substrate was added and the enzymatic rate was observed. For steady-state kinetic assays, the substrate concentration was varied, and data was fit to the equation:v = V max[S]/(K m + [S]). The rate of DTT oxidation catalyzed by AhpC or AhpD in the presence of the peroxide substrate was measured by monitoring the change in absorbance at 310 nm due to formation of the DTT disulfide (32.Iyer K.S. Klee W.A. J. Biol. Chem. 1973; 248: 707-710Abstract Full Text PDF PubMed Google Scholar). A Cary 1E spectrophotometer was used to obtain this data. The buffer and the water used for the assays were Chelex-pretreated as recommended by the supplier. Typical conditions for the assays were: 100 mm KPi, pH 7.0, 1 mm EDTA, and 10 mm DTT in a 1-ml quartz cuvette at 25 °C (maintained with a circulating water bath). The initial rate of DTT oxidation was obtained by calculating the slope over the first 11 s after addition and mixing of the peroxide. The initial rates were corrected for the background oxidation of DTT by the peroxides in the absence of the enzyme. A 50-μl solution containing an equimolar mixture (0.23 μmol) of cumene hydroperoxide and either AhpC or AhpD was equilibrated in KPi, pH 7.0, buffer for 120 min. The reaction was then quenched by addition of an equal volume (50 μl) of a solution of 6% acetic acid in acetonitrile. The protein that precipitated was removed by centrifugation, and the supernatant was injected onto a Hewlett-Packard 1090 HPLC system equipped with an Axxiom ODS (4.6 × 250 mm) reverse-phase HPLC column. The products were separated using 20% acetonitrile and 80% water at a flow rate of 1 ml/min. The detector was set at 260 nm. Control reactions were performed in the same buffer (50 mm KPi, pH 7.0, 100 mm KCl, 0.1 mm EDTA, and 5% glycerol) but without the enzyme. Product peaks were identified by comparison with authentic standards under identical elution conditions. Hydroperoxides were generated using the Schenck reaction of singlet oxygen with unactivated olefins bearing allylic hydrogens (33.Prein M. Adam W. Agnew. Chem. Int. Ed. Eng. 1996; 35: 477-494Crossref Scopus (283) Google Scholar,34.Wasserman H.H. Ives J.L. Tetrahedron. 1980; 37: 1825-1852Crossref Scopus (208) Google Scholar). In general, a solution of the olefin (3 mmol) and the sensitizer tetraphenylporphine (14.8 mg, 10 mm) in 25 ml of CCl4 was irradiated with a Sylvania 750 W lamp at 0 °C. A slow stream of oxygen was bubbled through the stirred solution for 5–6 h. The solvent was then removed in vacuo, and the hydroperoxide products were purified by column chromatography followed by crystallization or distillation, as appropriate. The products were identified by comparison of their physical properties and spectra with those in the literature and/or by reduction to the corresponding alcohols with triphenylphosphine (not shown). The structures of the hydroperoxide products are shown in Fig.1. 5-Hydroperoxy-6-methyl-6-hepten-2-one (1) and 6-hydroperoxy-6-methyl-4-hepten-2-one (2) were obtained from the oxidation of 6-methyl-5-hepten-2-one in a 2:1 ratio, respectively, and in 96% yield: colorless oil, bp 68–71 (∼5 mm Hg),R f = 0.59 (silica gel, 1:10 ethyl acetate:hexane); IR (KBr) 3439 (-OOH) and 1678 cm−1 (C=O); 1H NMR (400 MHz): (1) 1.36 (s, 3H, CH3), 1.70 (m, 2H, CH2), 1.73 (s, 3H, CH3), 1.92 (m, 2H, CH2), 4.37 (d, 1H, 3 J HH= 11.1 Hz, CHOOH), 4.91 (s, 1H, =CH2), and 4.94 ppm (s, 1H, =CH2); (2) 1.24 (s, 3H, CH3), 1.33 (s, 3H, CH3), 2.22 (s, 3H, CH3), 3.28 (d, 2H,3 J HH = 6.5 Hz, CH2), 6.30 (d, 1H, 3 J HH = 16.0 Hz, CH), and 6.58 ppm (dd, 1H, 3 J HH= 16.0, and 3 J HH = 6.6 Hz, CH). Trans-pinocarveylhydroperoxide (3) was obtained in 89% yield by irradiation of α-pinene as described by Schencket al. (35.Schenck G.O. Eggert H. Denk W. Justus Liebigs Ann. Chem. 1953; 584: 177-198Crossref Scopus (95) Google Scholar) and subsequently Capdeville and Maumy (36.Capdeville P. Maumy T.H.L. Tetrahedron Lett. 1980; 21: 2417-2420Crossref Scopus (14) Google Scholar): yellow oil, R F = 0.59 (benzene); IR (KBR) 3395 cm−1 (OOH); EIMS m/z 168;1H NMR (400 MHz): 0.66 (s, 3H, CH3), 1.26 (s, 3H, CH3), 1.48 (d, 1H,3 J HH = 9.8 Hz, CH), 1.93 (m, 2H, CH2), 2.23 (m, 1H, CH2), 2.32 (m, 1H, CH2), 2.47 (m, 1H, CH), 4.61 (d, 1H,3 J HH = 8.2 Hz, CHOOH), 4.99 (s, 1H, =CH2), 5.12 (s, 1H, =CH2), and 7.97 ppm (s, 1H, OOH). 7α-Hydroperoxy-3β-hydroxycholest-6-ene (6) resulted from the photosenzitized oxidation of cholesterol as reported by Beckwith et al. (37.Beckwith A.L.J. Davies A.G. Davison I.G.E. Maccoll A. Mruzek M.H. J. Chem. Soc. Perkin Trans. 1989; II: 815-824Crossref Scopus (103) Google Scholar). It was obtained in 32% yield as a white solid (recrystallization from benzene) m.p. 157–158 °C (literature 152–153 °C (37.Beckwith A.L.J. Davies A.G. Davison I.G.E. Maccoll A. Mruzek M.H. J. Chem. Soc. Perkin Trans. 1989; II: 815-824Crossref Scopus (103) Google Scholar), 154–156.5 °C (38.Nickon A. Bagli J.F. J. Am. Chem. Soc. 1961; 83: 1498-1508Crossref Scopus (141) Google Scholar)), R F = 0.69 (silica gel, 1:1 benzene:ethyl acetate): IR (KBr) 3335 cm−1 (-OOH);1H NMR (400 MHz): 0.65 (s, 3H, CH3), 0.85 (d, 3H, 3 J HH = 6.6 Hz, CH3), 0.90 (d, 3H,3 J HH = 6.3 Hz, CH3), 0.98 (s, 3H, CH3), 1.11–1.39 (m, 8H), 1.48 (m, 6H), 1.54 (m, 4H), 1.57 (m, 2H), 1.86 (m, 3H), 1.97 (d, 2H,3 J HH = 12.6 Hz, CH2), 2.37 (m, 2H), 3.61 (m, 1H, CHOH), 4.15 (t, 1H,3 J HH = 4.0 Hz, CHOOH), and 5.71 ppm (dd, 1H, 3 J HH = 4.8 and4 J HH = 1.6 Hz, =CH-). Trans-9-hydroperoxyoctadec-10-enoic acid (4) andtrans-10-hydroperoxyoctadec-8-enoic acid (5) were obtained in a ratio of 1:0.8, respectively, from oleic acid as reported by Porter and Wujek (39.Porter N.A. Wujek J.S. J. Org. Chem. 1987; 52: 5085-5089Crossref Scopus (78) Google Scholar): yellow oil obtained in 60% yield,R F = 0.33 (silica gel, ethyl acetate). An additional purification step was required for these compounds using C18 reverse phase HPLC, with a 30–78% acetonitrile gradient over 30 min with a flow rate of 1 ml/min. 1H NMR (400 MHz): (4) 0.89 (m, 3H, CH3), 1.28 (broad peak, 18H), 1.63 (m, 2H, CH2), 2.08 (m, 2H, CH2), 2.35 (m, 2H, CH2), 4.27 (q, 1H, 3 J HH= 7.8 Hz, CHOOH), 5.36 (dd, 1H,3 J HH = 8.0 and3 J HH = 15.0 Hz, CH), and 5.76 ppm (dt, 1H, 3 J HH = 6.6 and3 J HH = 15.1 Hz, CH) (4 +5). 13C NMR: (4) δ = 179.9 (COOH), δ11 = 137.2, δ10 = 128.4, δ9 = 87.1; (5) δ = 179.5 (COOH), δ8 = 136.7, δ9 = 128.8, δ10 = 87.1. Both proteins were independently overexpressed in E. coli strain BL21(DE3), using a pET vector for AhpC and a pACYC vector for AhpD. Lower yields or proteolyzed proteins were obtained when a pET23a or pUC19 vector was used, or when efforts were made to express the protein in the DH5α or XL1-BLUE strains of E. coli. Large quantities of AhpD (>50%) were lost as insoluble inclusion bodies when isopropyl-β-d-thiogalactopyranoside-induced expression was performed above 25 °C. Expressions were therefore carried out at a lower temperature to minimize this problem, although some loss of protein still occurred. Attempts to refold the precipitated protein were unsuccessful. In contrast, AhpC was produced in high amounts, and no inclusion bodies were observed even when the protein was expressed at 37 °C. A single ion-exchange chromatographic protocol was sufficient to purify the two enzymes. Both proteins were judged to be >95% pure by denaturing SDS-polyacrylamide gel electrophoresis (Fig. 2). Due to the absence of a strong chromophore in either protein, enzyme concentrations were determined from the molar absorption coefficients using the method of Pace et al. (40.Pace C.N. Vajdos F. Fee L. Grimsley G. Gray T. Protein Sci. 1995; 4: 2411-2423Crossref PubMed Scopus (3472) Google Scholar). For AhpC, the calculated ε (280) is 25,170 m−1cm−1, and for AhpD, the calculated ε (280) is 15,720m−1 cm−1. Size exclusion chromatography indicated that AhpC was primarily present as a higher-order oligomer (10–12 mer) (Fig.3). A minor peak at approximately 15 min in the AhpC chromatogram indicated that a small amount of the dimer was also present. This dimer could represent either modified protein that is unable to oligomerize or the fraction of the protein present as the dimer in a dimer-oligomer equilibrium. A dimeric rather than oligomeric species was observed for AhpD
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