The Mechanism of Mycobacterium tuberculosis Alkylhydroperoxidase AhpD as Defined by Mutagenesis, Crystallography, and Kinetics-论文阅读讨论-ReadPaper - 轻松读论文 | 专业翻译 | 一键引文

DOI: 10.1074/jbc.m303747200

CAS-2 JCR-Q2 SCIE EI

Aleksey KoshkinChristine M. NunnSnezana DjordjevicPaul R. Ortiz de Montellano

Journal of Biological Chemistry

Aug 2003

39被引用

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摘要原文

AhpD, a protein with two cysteine residues, is required for physiological reduction of the Mycobacterium tuberculosis alkylhydroperoxidase AhpC. AhpD also has an alkylhydroperoxidase activity of its own. The AhpC/AhpD system provides critical antioxidant protection, particularly in the absence of the catalase-peroxidase KatG, which is suppressed in most isoniazid-resistant strains. Based on the crystal structure, we proposed recently a catalytic mechanism for AhpD involving a proton relay in which the Glu118 carboxylate group, via His137 and a water molecule, deprotonates the catalytic residue Cys133 (Nunn, C. M., Djordjevic, S., Hillas, P. J., Nishida, C., and Ortiz de Montellano, P. R. (2002) J. Biol. Chem. 277, 20033–20040). A possible role for His132 in subsequent formation of the Cys133-Cys130 disulfide bond was also noted. To test this proposed mechanism, we have expressed the H137F, H137Q, H132F, H132Q, E118F, E118Q, C133S, and C130S mutants of AhpD, determined the crystal structures of the H137F and H132Q mutants, estimated the pK a values of the cysteine residues, and defined the kinetic properties of the mutant proteins. The collective results strongly support the proposed catalytic mechanism for AhpD. AhpD, a protein with two cysteine residues, is required for physiological reduction of the Mycobacterium tuberculosis alkylhydroperoxidase AhpC. AhpD also has an alkylhydroperoxidase activity of its own. The AhpC/AhpD system provides critical antioxidant protection, particularly in the absence of the catalase-peroxidase KatG, which is suppressed in most isoniazid-resistant strains. Based on the crystal structure, we proposed recently a catalytic mechanism for AhpD involving a proton relay in which the Glu118 carboxylate group, via His137 and a water molecule, deprotonates the catalytic residue Cys133 (Nunn, C. M., Djordjevic, S., Hillas, P. J., Nishida, C., and Ortiz de Montellano, P. R. (2002) J. Biol. Chem. 277, 20033–20040). A possible role for His132 in subsequent formation of the Cys133-Cys130 disulfide bond was also noted. To test this proposed mechanism, we have expressed the H137F, H137Q, H132F, H132Q, E118F, E118Q, C133S, and C130S mutants of AhpD, determined the crystal structures of the H137F and H132Q mutants, estimated the pK a values of the cysteine residues, and defined the kinetic properties of the mutant proteins. The collective results strongly support the proposed catalytic mechanism for AhpD. Mycobacterium tuberculosis infects an estimated 8 million per year and kills 2–3 million people in the same time span (1Dye C. Scheele S. Dolin P. Pathania V. Raviglione M.C. J. Am. Med. Assoc. 1999; 282: 677-686Crossref PubMed Scopus (2729) Google Scholar). It is estimated that ∼2 billion people have been exposed to this lethal pathogenic organism and thus are at risk of developing the active disease. The majority of infected individuals reside in the third world, but the rates of infection in other areas that are undergoing rapid social change, such as the Soviet Union, are increasing at an alarming rate (2Keshavjee S. Becerra M.C. J. Am. Med. Assoc. 2000; 283: 1201Crossref Scopus (29) Google Scholar). Furthermore, partially as a result of the symbiotic relationship between human immunodeficiency virus and tuberculosis, the incidence of multidrug-resistant tuberculosis is rapidly increasing (3Pablos-Méndez A. Rafiglione M.C. Laszlo A. Binkin N. Rieder H.L. Bustreo F. Cohn D.L. Labregts-Van Weezenbeek C.S.B. Kim S.J. Chaulet P. Nunn P. N. Engl. J. Med. 1998; 338: 1641-1649Crossref PubMed Scopus (834) Google Scholar). The resurgence of tuberculosis as a world-wide phenomenon, in conjunction with the recent determination of the M. tuberculosis genome, has fuelled a renewed search for agents that are active against drug-resistant strains, completely sterilize the infection, and/or shorten the duration of drug therapy and thus promote drug compliance. Mutations in the M. tuberculosis catalase-peroxidase KatG result in resistance to the prodrug isoniazid, because KatG is required to oxidize this drug to its biologically active form (4Zhang Y. Dhandayuthapani S. Deretic V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13212-13216Crossref PubMed Scopus (100) Google Scholar, 5Slayden R.A. Barry III, C.E. Microbes Infect. 2000; 2: 659-669Crossref PubMed Scopus (169) Google Scholar, 6Zhang Y. Heym B. Allen B. Young D. Cole S.T. Nature. 1992; 358: 591-593Crossref PubMed Scopus (1097) Google Scholar, 7Johnson K. Schultz P.G. J. Am. Chem. Soc. 1994; 116: 7425-7426Crossref Scopus (271) Google Scholar, 8Wengenack N.L. Rusnak F. Biochemistry. 2001; 40: 8990-8996Crossref PubMed Scopus (82) Google Scholar, 9Rozwarski D.A. Grant G.A. Barton D.H.R. Jacobs Jr., W.R. Sacchettini J.C. Science. 1998; 279: 98-102Crossref PubMed Scopus (618) Google Scholar). In a similar manner, mutations in the flavoprotein monooxygenase EtaA result in resistance to ethionamide, because it is also a prodrug that must be oxidized by EtaA to its active form (10DeBarber A.E. Mdluli K. Bosman M. Bekker L.-G. Barry III, C.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9677-9682Crossref PubMed Scopus (307) Google Scholar, 11Baulard A.R. Betts J.C. Engohang-Ndong J. Quan S. Brennan P.J. Locht C. Besra G.S. J. Biol. Chem. 2000; 275: 28326-28331Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). Interestingly, even though the activating enzymes differ, the mutations of KatG and EtaA that cause resistance to isoniazid and ethionamide, respectively, result in elevated expression of AhpC (4Zhang Y. Dhandayuthapani S. Deretic V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13212-13216Crossref PubMed Scopus (100) Google Scholar, 12Sherman D.R. Mdlui 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, 13Sherman 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, 14Chae 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, 15Deretic V. Pagan-Ramos E. Zhang Y. Dhandayuthapani S. Via L.E. Nat. Biotechnol. 1996; 14: 1557-1561Crossref PubMed Scopus (52) Google Scholar, 17Wilson M. DeRisi J. Kristensen H.-H. Imboden P. Rane S. Brown P.O. Schoolnik G.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12833-12838Crossref PubMed Scopus (497) Google Scholar). AhpC is thought to provide protection against the oxidative stress associated with both of these mutations, particularly against loss of the KatG peroxidase activity (12Sherman D.R. Mdlui 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 is a member of the ubiquitous peroxiredoxin family. In the peroxiredoxins, a cysteine residue reacts with a peroxide or other oxidant to give a sulfenic acid (-SOH) intermediate (19Poole L.B. Ellis H.R. Methods Enzymol. 2002; 348: 122-136Crossref PubMed Scopus (80) Google Scholar, 20Hoffman B. Hecht H.-J. Flohé L. Biol. Chem. 2002; 383: 347-364PubMed Google Scholar). This sulfenic acid is then converted to a disulfide bond by intramolecular or intermolecular reaction with a second sulfhydryl group. Finally, the disulfide bond is reduced to regenerate the cysteine thiol groups. The disulfide bond is reduced by different mechanisms in different organisms; in yeast, the reduction is mediated by thioredoxin and thioredoxin reductase (21Chae H.Z. Chung S.J. Rhee S.G. J. Biol. Chem. 1994; 269: 27670-27678Abstract Full Text PDF PubMed Google Scholar) and in Salmonella typhimurium by a flavoprotein known as AphF (22Ellis H.R. Poole L.B. Biochemistry. 1997; 36: 13357-13364Crossref PubMed Scopus (47) Google Scholar). The M. tuberculosis thioredoxin and thioredoxin reductase, however, do not reduce the corresponding AhpC (23Zhang Z. Hillas P.J. Ortiz de Montellano P.R. Arch. Biochem. Biophys. 1999; 363: 19-26Crossref PubMed Scopus (38) Google Scholar), and no hom*ologue of AhpF is detected in the genome of this organism by BLAST searches. However, a gene (Rv2429) coding for AhpD, a protein with no sequence identity to AhpC or AhpF, is located immediately adjacent to the AhpC gene. Recent work has shown that AhpD functions as the reducing partner for AhpC. AhpD itself is reduced by a novel system consisting of dihydrolipoamide succinyltransferase (SucB), a lipoamide-containing protein, and dihydrolipoamide dehydrogenase (25Bryk R. Lima C.D. Erdjument-Bromage H. Tempst P. Nathan C. Science. 2002; 295: 1073-1077Crossref PubMed Scopus (328) Google Scholar). SucB can be replaced in this system by dihydrolipoamide itself. Interestingly, AhpD, in addition to being the reducing partner for AhpC, has independent alkylhydroperoxidase activity of its own when AhpF from S. typhimurium is used as a surrogate reducing partner (24Hillas P.J. Soto del Alba F. Oyarzabal J. Wilks A. Ortiz de Montellano P.R. J. Biol. Chem. 2000; 275: 18801-18809Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The crystal structure of AhpD was independently determined by two laboratories (25Bryk R. Lima C.D. Erdjument-Bromage H. Tempst P. Nathan C. Science. 2002; 295: 1073-1077Crossref PubMed Scopus (328) Google Scholar, 26Nunn C.M. Djordjevic S. Hillas P.J. Nishida C. Ortiz de Montellano P.R. J. Biol. Chem. 2002; 277: 20033-20040Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). AhpD is a hom*otrimeric protein in which the individual subunits consist of 177 amino acids with a molecular mass of 18,781 Da (25Bryk R. Lima C.D. Erdjument-Bromage H. Tempst P. Nathan C. Science. 2002; 295: 1073-1077Crossref PubMed Scopus (328) Google Scholar, 26Nunn C.M. Djordjevic S. Hillas P.J. Nishida C. Ortiz de Montellano P.R. J. Biol. Chem. 2002; 277: 20033-20040Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Two cysteines, Cys130 and Cys133, are present in the AhpD sequence (numbering based on inclusion of the initial methionine) in a Cys-His-Ser-Cys motif within a novel protein fold. Site-specific mutagenesis of the cysteines has shown that both cysteines, but particularly Cys133, are critical for the alkylhydroperoxidase activity supported by the S. typhimurium AhpF (24Hillas P.J. Soto del Alba F. Oyarzabal J. Wilks A. Ortiz de Montellano P.R. J. Biol. Chem. 2000; 275: 18801-18809Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 25Bryk R. Lima C.D. Erdjument-Bromage H. Tempst P. Nathan C. Science. 2002; 295: 1073-1077Crossref PubMed Scopus (328) Google Scholar). Despite the absence of structural hom*ology to other proteins, analysis of the detailed structure of AhpD identified several residues that could be involved in the catalytic activity of the enzyme (26Nunn C.M. Djordjevic S. Hillas P.J. Nishida C. Ortiz de Montellano P.R. J. Biol. Chem. 2002; 277: 20033-20040Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) based on local structural analogy to the active site of a functional analogue, thioredoxin. Specifically, the carboxylate anion of Glu118 is 2.6 Å from the nitrogen of His137 and is hydrogen-bonded to it (Fig. 1). His137 is hydrogen-bonded to a water molecule 2.5 Å away that, in turn, interacts with Cys133 at a distance of 3.3 Å. These hydrogen bonding interactions provide a reasonable mechanism for deprotonation of Cys133, the residue thought to react with the O–O bond of peroxides or the disulfide bond present in the oxidized form of AhpC. The location of His132 4.8 Å from Cys130 and 3.7 Å from Cys133 in the reduced enzyme suggested that it might also play a role in the catalytic mechanism. These mechanistic proposals have been tested in the present study by mutating the residues, determining the crystal structures of two of the mutants, and determining the effects of the mutations on the catalytic activities in both the AhpF and AhpD/AhpC/lipoamide/dihydrolipoamide dehydrogenase assay systems. Materials—All chemical reagents were purchased from Sigma. Escherichia coli strain BL21(DE3) was from Novagen, and strain DH5α was from Invitrogen. Q-Sepharose Fast Flow was purchased from Amersham Biosciences, and polyethyleneimine was from Research Biotechnologies, Inc. (Natick, MA). LB medium was obtained from Invitrogen. Chloramphenicol, NaCl, NaOH, SDS, and MOPS 1The abbreviation used is: MOPS, 4-morpholinepropanesulfonic acid. were from Fisher. Protein molecular weight standards were from Invitrogen. Purified proteins were concentrated using Millipore YM10 regenerated cellulose ultrafiltration membranes. Isopropyl-1-thio-β-d-galactopyranoside was from Promega (Madison, WI), and polyethyleneimine (10% solution) was from Research Biochemicals International (Natick, MA). Lipoamide, D,L-6,8-thioctic acid amide, and dihydrolipoamide dehydrogenase (EC 1.8.1.4) from bovine intestinal mucosa, 100 units/mg protein were purchased from Sigma. Oligonucleotide synthesis and DNA sequencing were performed by the Biomolecular Resource Center of the University of California, San Francisco, CA. A PerkinElmer Life Sciences 480 DNA thermal cycler was used for PCR experiments. An Amersham Biosciences Sephadex 200 column, connected to an Amersham Biosciences PCC-500 fast performance liquid chromatography system, was used for the final stage of protein purification. A Hewlett-Packard HP-8452 UV-visible spectrophotometer was used for all spectroscopic measurements. S. typhimurium AhpF was generously provided by Patrick Hillas, as were the C130S and C133S constructs (24Hillas P.J. Soto del Alba F. Oyarzabal J. Wilks A. Ortiz de Montellano P.R. J. Biol. Chem. 2000; 275: 18801-18809Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). All expression plasmids were introduced into competent BL21(DE3) E. coli cells. AhpD Mutagenesis—The single mutants of AhpD were made using the QuikChange site-directed mutagenesis kit from Stratagene (La Jolla, CA) as described previously (24Hillas P.J. Soto del Alba F. Oyarzabal J. Wilks A. Ortiz de Montellano P.R. J. Biol. Chem. 2000; 275: 18801-18809Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The mutations were confirmed by DNA sequencing. The primers used for the mutagenesis were as follows: AhpDE118F (forward primer, 5′-AAA GCC AAC TTC TTT CTC TGG TCC TTC-3′ and reverse primer, 5′-GAA GGA CCA GAG AAA GAA GTT GGC TTT-3′), AhpDE118Q (forward primer, 5′-AAA GCC AAC TTC CAG CTC TGG TCC TTC-3′ and reverse primer, 5′-GAA GGA CCA GAG CTG GAA GTT GGC TTT-3′), AhpDH137Q (forward primer, 5′-TGC CTC GTC GCC CAA GAG CAC ACG CTG-3′ and reverse primer, 5′-CAG CGT GTG CTC TTG GGC GAC GAG GCA-3′), AhpDH137F (forward primer, 5′-TGC CTC GTC GCC TTC GAG CAC ACG CTG-3′ and reverse primer, 5′-CAG CGT GTG CTC GAA GGC GAC GAG GCA-3′), AhpDH132Q (forward primer, 5′-AAC GGG TGC TCG CAA TGC CTC GTC GCC-3′ and reverse primer, 5′-GGC GAC GAG GCA TTG CGA GCA CCC GTT-3′), AhpDH132F (forward primer, 5′-AAC GGG TGC TCG TTT TGC CTC GTC GCC-3′ and reverse primer, 5′-GGC GAC GAG GCA AAA CGA GCA CCC GTT-3′). The underlined codons represent the mutation. Bacterial Cell Growth—Bacterial growth was carried out at 37 °C in LB medium containing 50 μg/ml chloramphenicol. One colony was used to inoculate 50 ml of LB medium containing the antibiotic, and the culture was incubated for 12 h. The culture was used to inoculate a 1-liter culture of LB containing the antibiotic at a ratio of 10 ml/liter. When the A 600 value of the culture reached 0.7–0.8, isopropyl-β-d-thiogalactopyranoside was added to a final concentration of 0.2 mm. Incubation was continued for 12 h at 20 °C. Cells were harvested by centrifugation at 5000 × g for 45 min at 4 °C and stored at –20 °C overnight. Protein Purification—M. tuberculosis AhpD and mutants were expressed heterologously in E. coli and purified according to the protocol of Hillas et al. (24Hillas P.J. Soto del Alba F. Oyarzabal J. Wilks A. Ortiz de Montellano P.R. J. Biol. Chem. 2000; 275: 18801-18809Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) with some modifications. Cells were suspended in a 6-fold excess (with respect to the initial weight of cells) of lysis buffer. The solution was stirred for 60 min at 4 °C. A polyethyleneimine supernatant was prepared and loaded onto the Q-Sepharose column. After loading, the resin was washed with the same buffer for 20 column volumes. The protein was eluted with a gradient from 0 to 0.75 m KCl in 50 mm potassium Pi, pH 7.0, 1.0 mm dithiothreitol, 1.0 mm EDTA, and 5% glycerol. The protein eluted at ∼0.2 m KCl. Fractions containing maximum amount of protein, as assessed by denaturing 20% polyacrylamide gels using the PhastSystem (Amersham Biosciences), were pooled and concentrated to 5 mg/ml or greater using a Millipore concentrator equipped with a YM10 (10,000 molecular weight cut-off) regenerated cellulose ultrafiltration membrane. Enzyme concentrations were determined from the molar absorption coefficients using the method of Pace et al. (27Pace C.N. Vadjos F. Fee L. Grimsley G. Gray T. Protein Sci. 1995; 4: 2411-2423Crossref PubMed Scopus (3470) Google Scholar) and a molar extinction coefficient of 15,720 m–1 cm–1. In contrast to AhpD a single ion-exchange chromatographic protocol was insufficient to purify the AhpD E118Q and H137Q mutants. Further purification by gel filtration on Superdex 200 using 50 mm MOPS, pH 7.2, 100 mm KCl, 20% glycerol, 5 mm dithiothreitol, and 0.2 mm EDTA was therefore carried out with all proteins. The proteins were judged to be >95% pure by denaturing SDS-PAGE. The proteins were finally aliquoted into 25- or 100-μl volumes, frozen on dry ice, and stored at –80 °C until used. AhpF-dependent Activity Assays—Rates of hydroperoxide reduction were determined anaerobically in a coupled assay with AhpF, monitoring the decrease in absorbance at 340 nm because of NADH oxidation as reported previously (24Hillas P.J. Soto del Alba F. Oyarzabal J. Wilks A. Ortiz de Montellano P.R. J. Biol. Chem. 2000; 275: 18801-18809Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The assays typically contained 2 mm hydroperoxide substrate in 100 mm potassium Pi, pH 7.0, 1 mm EDTA, 0.25 mm NADH, 20 μm AhpD or mutant, and 10 μm AhpF. Background NADH oxidation caused by AhpF was monitored, the hydroperoxide substrate was added, and the enzymatic rate was observed. AhpD-dependent Peroxidase Activity AhpC Assays—The rate of NADH oxidation catalyzed by AhpC in the presence of AhpD, lipoamide, and bovine lipoamide dehydrogenase was measured as described by monitoring the change in absorbance at 340 nm (25Bryk R. Lima C.D. Erdjument-Bromage H. Tempst P. Nathan C. Science. 2002; 295: 1073-1077Crossref PubMed Scopus (328) Google Scholar). Typical conditions for the assays were as follows: 50 mm potassium Pi, pH 7.0, 1 mm EDTA, 200 μm NADH, 2.5 μm AhpC, 2.5 μm AhpD, 0.2 units of bovine dihydrolipoamide dehydrogenase, and 50 μm lipoamide. This assay can be used to evaluate the activity of AhpD and AhpD mutants, because the AhpD-supported activity of AhpC directly depends on the activity of AhpD. For steady-state kinetic assays, the substrate concentration was varied, and data were fit to the equation v = V max [S]/(K m + [S]). Measurement of Thiol pK a Values by UV Absorption—The pH-dependent ionization of the cysteine thiols was monitored by the absorbance of the thiolate anion at 240 nm as described (28Nelson J.W. Creighton T.E. Biochemistry. 1994; 33: 5974-5983Crossref PubMed Scopus (225) Google Scholar, 29Reckenfelderbäumer N. Krauth-Siegel L. J. Biol. Chem. 2002; 277: 17548-17555Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). All of the measurements were carried out at 25 °C, with 10 μm AhpD or AhpD mutant in 1 mm each of argon-purged citrate, borate, and phosphate buffer containing 0.2 mm KCl and 5 mm dithiothreitol. The solution was titrated with 0.2 m KOH. Crystallization—Crystallization of the AhpD H137F and H132Q mutants was carried out by the hanging drop vapor diffusion method reported previously (26Nunn C.M. Djordjevic S. Hillas P.J. Nishida C. Ortiz de Montellano P.R. J. Biol. Chem. 2002; 277: 20033-20040Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Hanging drops were prepared by mixing equal volumes of protein solution (4.5 mg/ml in 25 mm MOPS buffer, pH 7.2, containing 50 mm KCL, 10% glycerol, 0.1 mm EDTA, and 5.0 mm dithiothreitol) with a reservoir solution containing 100 mm sodium citrate buffer at pH 5.6 containing 200 mm ammonium acetate and 26% polyethylene glycol 4000. Crystals grew as rhombohedral prisms over a period of days. The crystals belong to space group C2 for both mutant structures with cell dimensions a = 96.60 Å, b = 62.18 Å, c = 89.48 Å, β = 121.81° for H137F and a = 100.12 Å, b = 58.65 Å, c = 88.91 Å, β = 120.47° for the H132Q mutant. A Matthews coefficient of 1.96 corresponds to 37% (v/v) solvent for the crystallographic asymmetric unit of H137F, and a value of 1.93 corresponds to 34% (v/v) solvent in the crystallographic asymmetric unit of H132Q (30Matthews B.W. J. Mol. Biol. 1968; 33: 491-497Crossref PubMed Scopus (7926) Google Scholar). Data Collection and Processing—Data collection parameters are shown in Table I. All data processing, integration, and scaling was carried out using HKL (31Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38609) Google Scholar) and the CCP4 suite of programs (32Collaborative Computational Project Number 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar).Table IData collection parametersH137F mutantH132Q mutantX-ray sourceStation 9.6 SRSStation 9.6 SRSWavelength (Å)0.870.87DetectorADSC Quantum-4 CCDADSC Quantum-4 CCDTemperature (K)110110Resolution range (Å)aNumbers in parentheses are for last shell.50-2.3 (2.38-2.3)50-2.3 (2.38-2.3)Total number of reflections396,017151,675Unique reflections18,39616,445Completeness (%)aNumbers in parentheses are for last shell.92.5 (70.3)95.1 (94.9)R mergebR merge = [Σhk1Σi | Ihk1 - <Ihk1> |/Σhk1 Σi |I|].0.11 (0.24)0.097 (0.12)a Numbers in parentheses are for last shell.b R merge = [Σhk1Σi | Ihk1 - <Ihk1> |/Σhk1 Σi |I|]. Open table in a new tab Model Building and Refinement—Throughout the refinement 10% of the reflections were used for cross-validation analysis (33Brunger A.T. Nature. 1992; 355: 472-475Crossref PubMed Scopus (3872) Google Scholar), and the behavior of R free was used to monitor the refinement strategy. All refinement was carried out using the program CNS v.1.1 (34Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.-S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar), and model building was carried out using programs O (35Jones T.A. Zou J.Y. Cowans S.W. Kjelgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar) and TURBOFRODO (36Rousel A. Inisan A.G. Knoops-Mouthuy E. Cambillau C. TURBO Manual. AFMB-IFRC1, Marseille, France2000Google Scholar). A protein trimer exists within the crystallographic asymmetric unit. One AhpD trimer from the native crystal structure (Protein Data Bank code 1GU9) was used as a model for molecular replacement. Cross-rotation and translation functions were calculated using data from 15 to 4 Å resolution, and a clear solution was obtained for both mutant structures. Following molecular replacement rigid body refinement was carried out prior to full positional refinement using simulated annealing with torsion angle dynamics, anisotropic scaling, energy minimization, individual isotropic B-factor refinement, and bulk-solvent correction against the maximum-likelihood target. Refinement progressed well with further rounds of positional and individual B factor refinement. Toward the end of the structure refinement electron density maps were used to locate and include solvent water within the refinement. Refinement parameters for both mutant structures are given in Table II.Table IIRefinement parametersH137F mutantH132Q mutantSpace groupC2C2Cell dimensions (Å)96.603 × 62.184 × 89.481, β = 121.81100.119 × 58.652 × 88.914, β = 120.47Resolution range (Å)50 - 2.350 - 2.4No. of protein atomsProtein A (residues 3 to 177) 1307Protein A (residues 3 to 175) 1289Protein B (residues 3 to 175) 1289Protein B (residues 3 to 175) 1289Protein C (residues 3 to 174) 1288Protein C (residues 3 to 171) 1265No. of solvent water molecules364370R cryst (%)18.422.3R free (%)28.635.0Root mean square deviation bonds (Å)0.0080.008Root mean square deviation angles (°)1.301.39Average B values (Å2)44.145.1Ramachandran plot90.7% - most favored regions83.1% - most favored regions8.0% - allowed regions13.6% - allowed regions Open table in a new tab Preparation of Figures—The structural figures were prepared using DINO (www.dino3d.org). Coordinates—The atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession codes 1LW1 (H137F) and 1ME5 (H132Q). Catalytic Activities of AhpD Mutant Proteins—The recently determined crystal structure of AhpD suggests important roles for Glu118, Cys130, Cys133, His132, and His137 in the catalytic mechanism of AhpD (25Bryk R. Lima C.D. Erdjument-Bromage H. Tempst P. Nathan C. Science. 2002; 295: 1073-1077Crossref PubMed Scopus (328) Google Scholar, 26Nunn C.M. Djordjevic S. Hillas P.J. Nishida C. Ortiz de Montellano P.R. J. Biol. Chem. 2002; 277: 20033-20040Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). To ascertain the roles of these residues, single mutants were constructed in which each of the cysteines was mutated to a serine, and each of the two histidines and the glutamic acid was mutated to either a phenylalanine or a glutamine. The mutant proteins were expressed and purified by minor modifications of the protocol reported earlier for purification of wild-type AhpD (24Hillas P.J. Soto del Alba F. Oyarzabal J. Wilks A. Ortiz de Montellano P.R. J. Biol. Chem. 2000; 275: 18801-18809Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 26Nunn C.M. Djordjevic S. Hillas P.J. Nishida C. Ortiz de Montellano P.R. J. Biol. Chem. 2002; 277: 20033-20040Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The two AhpD Cys → Ser mutants and the two His → Phe mutants were expressed in yields comparable with those of the wild-type protein (∼25 mg/L), but the AhpD H137Q, E118F, and E118Q mutants were expressed at significantly lower levels (∼20% of wild-type). The glutamine mutants do not appear to bind to Q-Sepharose as strongly as native AhpD or the other mutants and tend to unfold and degrade relatively easily. The glutamine substitution may destabilize the protein fold or may force the protein into a conformation that exposes proteolytically sensitive sites. The catalytic activities of the mutants were evaluated in two assay systems. In the first system, the ability of the AhpD mutants to reduce cumene hydroperoxide in the presence of the S. typhimurium AhpF and NADH was evaluated (24Hillas P.J. Soto del Alba F. Oyarzabal J. Wilks A. Ortiz de Montellano P.R. J. Biol. Chem. 2000; 275: 18801-18809Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). This assay system tests the ability of AhpD to directly reduce a hydroperoxide in a reaction supported by a surrogate electron donor partner. The AhpF-supported catalytic activities of wild-type AhpD and its mutants are shown in Fig. 2. As reported previously (24Hillas P.J. Soto del Alba F. Oyarzabal J. Wilks A. Ortiz de Montellano P.R. J. Biol. Chem. 2000; 275: 18801-18809Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), the two cysteine mutations impair the alkyl peroxidase activity of AhpD, with mutation of Cys133 completely suppressing the activity and mutation of Cys130 decreasing it to less than 5% of the wild-type activity (Fig. 2). Likewise, the two His to Phe mutations decrease the catalytic activity, but mutation of His137 causes a greater decrease than mutation of His132. Mutation of the two His residues to glutamines also decreases the level of activity but to a lower extent than mutation to phenylalanines. As found for the phenylalanine mutants, the H137Q mutation decreased the activity more severely than the H132Q mutation. Replacement of Glu118 by a glutamine only modestly lowered the catalytic activity, whereas replacement by a phenylalanine decreased the activity to a much higher extent. In the second assay system, reduction of cumene hydroperoxide was measured in the fully reconstituted system consisting of AhpC, the AhpD mutant, lipoamide, bovine dihydrolipoamide dehydrogenase, and NADH (25Bryk R. Lima C.D. Erdjument-Bromage H. Tempst P. Nathan C. Science. 2002; 295: 1073-1077Crossref PubMed Scopus (328) Google Scholar). The lipoamide-dihydrolipoamide dehydrogenase-AhpC assay (Fig. 3) gives higher absolute alkylhydroperoxidase activities than the AhpF-dependent assay (Fig. 2). Nevertheless, as shown in Fig. 3, the impairment of the catalytic activities caused by the mutations follows the same trends as seen for direct reduction of the peroxide by AhpD/AhpF. The ratios among the activities of the different mutants are similar to those found in the AhpF assay. Thus, the two cysteine mutants are completely inactive in this assay, in agreement with an earlier report (25Bryk R. Lima C.D. Erdjument-Bromage H. Tempst P. Nathan C. Science. 2002; 295: 1073-1077Crossref PubMed Scopus (328) Google Scholar), the H137F mutation causes a great

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The Mechanism of Mycobacterium tuberculosis Alkylhydroperoxidase AhpD as Defined by Mutagenesis, Crystallography, and Kinetics-论文阅读讨论-ReadPaper - 轻松读论文 | 专业翻译 | 一键引文 | 图表同屏 (2024)