Characterization of Active Site Structure in CYP121: A Cytochrome P450 Essential for Viability of Mycobacterium Tuberculosis H37Rv*
2008; Elsevier BV; Volume: 283; Issue: 48 Linguagem: Inglês
10.1074/jbc.m802115200
ISSN1083-351X
AutoresKirsty J. McLean, Paul Carroll, Daniel Lewis, Adrian J. Dunford, Harriet E. Seward, Rajasekhar Neeli, Myles R. Cheesman, Laurent Marsollier, P. R. Douglas, W. Ewen Smith, Ida Rosenkrands, Stewart T. Cole, David Leys, Tanya Parish, Andrew W. Munro,
Tópico(s)Biochemical and Molecular Research
ResumoMycobacterium tuberculosis (Mtb) cytochrome P450 gene CYP121 is shown to be essential for viability of the bacterium in vitro by gene knock-out with complementation. Production of CYP121 protein in Mtb cells is demonstrated. Minimum inhibitory concentration values for azole drugs against Mtb H37Rv were determined, the rank order of which correlated well with Kd values for their binding to CYP121. Solution-state spectroscopic, kinetic, and thermodynamic studies and crystal structure determination for a series of CYP121 active site mutants provide further insights into structure and biophysical features of the enzyme. Pro346 was shown to control heme cofactor conformation, whereas Arg386 is a critical determinant of heme potential, with an unprecedented 280-mV increase in heme iron redox potential in a R386L mutant. A homologous Mtb redox partner system was reconstituted and transported electrons faster to CYP121 R386L than to wild type CYP121. Heme potential was not perturbed in a F338H mutant, suggesting that a proposed P450 superfamily-wide role for the phylogenetically conserved phenylalanine in heme thermodynamic regulation is unlikely. Collectively, data point to an important cellular role for CYP121 and highlight its potential as a novel Mtb drug target. Mycobacterium tuberculosis (Mtb) cytochrome P450 gene CYP121 is shown to be essential for viability of the bacterium in vitro by gene knock-out with complementation. Production of CYP121 protein in Mtb cells is demonstrated. Minimum inhibitory concentration values for azole drugs against Mtb H37Rv were determined, the rank order of which correlated well with Kd values for their binding to CYP121. Solution-state spectroscopic, kinetic, and thermodynamic studies and crystal structure determination for a series of CYP121 active site mutants provide further insights into structure and biophysical features of the enzyme. Pro346 was shown to control heme cofactor conformation, whereas Arg386 is a critical determinant of heme potential, with an unprecedented 280-mV increase in heme iron redox potential in a R386L mutant. A homologous Mtb redox partner system was reconstituted and transported electrons faster to CYP121 R386L than to wild type CYP121. Heme potential was not perturbed in a F338H mutant, suggesting that a proposed P450 superfamily-wide role for the phylogenetically conserved phenylalanine in heme thermodynamic regulation is unlikely. Collectively, data point to an important cellular role for CYP121 and highlight its potential as a novel Mtb drug target. The human pathogen Mycobacterium tuberculosis (Mtb) 4The abbreviations used are: MtbM. tuberculosisX-gal5-bromo-4-chloro-3-indolyl-?-d-galactopyranosideWTwild typePIMphenylimidazoleRRresonance Raman. 4The abbreviations used are: MtbM. tuberculosisX-gal5-bromo-4-chloro-3-indolyl-?-d-galactopyranosideWTwild typePIMphenylimidazoleRRresonance Raman. has made an alarming resurgence and again poses a global threat to human health (World Health Organization fact sheet on ?Tuberculosis?; located on the World Wide Web). The worldwide spread of tuberculosis has been fuelled by the development and spread of drug- and multidrug-resistant Mtb strains (2Matsumoto M. Hashizume H. Tsubouchi H. Sasaki H. Itotani M. Kuroda H. Tomishige T. Kawasaki M. Komatsu M. Curr. Top. Med. Chem. 2007; 7: 499-507Crossref PubMed Scopus (32) Google Scholar). The growing numbers of Mtb strains resistant to front line antitubercular drugs (e.g. rifampicin and isoniazid) has revealed a dearth of effective second line agents and has highlighted a desperate need for the development of novel drugs (3Spigelman M.K. J. Infect. Dis. 2007; 196: S28-S34Crossref PubMed Scopus (142) Google Scholar). M. tuberculosis 5-bromo-4-chloro-3-indolyl-?-d-galactopyranoside wild type phenylimidazole resonance Raman. M. tuberculosis 5-bromo-4-chloro-3-indolyl-?-d-galactopyranoside wild type phenylimidazole resonance Raman. Against this backdrop, the determination of the Mtb H37Rv genome sequence (and latterly the Mtb CDC1551 sequence) provided important new information toward a more detailed understanding of the biology of Mtb, its genome organization, and its protein repertoire (4Cole S.T. Brosch R. Parkhill J. Garnier T. Churcher C. Harris D. Gordon S.V. Eiglmeier K. Gas S. Barry III, C.E. Tekaia F. Badcock K. Basham D. Brown D. Chillingworth T. Connor R. Davies R. Devlin K. Feltwell T. Gentles S. 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 R. Squares S. Sulston J.E. Taylor K. Whitehead S. Barrell B.G. Nature. 1998; 393: 537-544Crossref PubMed Scopus (6504) Google Scholar, 5Fleischmann R.D. Alland D. Eisen J.A. Carpenter L. White O. Peterson J. DeBoy R. Dodson R. Gwinn M. Haft D. Hickey E. Kolonay J.F. Nelson W.C. Umayam L.A. Ermolaeva M. Salzberg S.L. Delcher A. Utterback T. Weidman J. Khouri H. Gill J. Mikula A. Bishai W. Jacobs Jr. Jacobs W.R. Venter J.C. Fraser C.M. J. Bacteriol. 2002; 184: 5479-5490Crossref PubMed Scopus (578) Google Scholar). Mtb H37Rv is a virulent strain that has been the most commonly used Mtb strain in laboratory and clinical studies for over 50 years. Its genome revealed an unexpectedly large number of genes encoding cytochrome P450 (CYP or P450) enzymes, 20 CYP genes in the 4.41-megabase Mtb H37Rv genome (4Cole S.T. Brosch R. Parkhill J. Garnier T. Churcher C. Harris D. Gordon S.V. Eiglmeier K. Gas S. Barry III, C.E. Tekaia F. Badcock K. Basham D. Brown D. Chillingworth T. Connor R. Davies R. Devlin K. Feltwell T. Gentles S. 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 R. Squares S. Sulston J.E. Taylor K. Whitehead S. Barrell B.G. Nature. 1998; 393: 537-544Crossref PubMed Scopus (6504) Google Scholar). The Escherichia coli genome (of similar size) is devoid of P450s, and the 57 P450s in the human genome are contained within a ~3000-megabase genome. Thus, the CYP gene ?density? in the Mtb genome is >200-fold that in the human genome, indicating important cellular roles for these Mtb oxygenases (6McLean K.J. Clift D. Lewis D.G. Sabri M. Balding P.R. Sutcliffe M.J. Leys D. Munro A.W. Trends Microbiol. 2006; 14: 220-228Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). In addition, the first prokaryotic example of a sterol demethylase P450 (CYP51B1) was identified in Mtb (4Cole S.T. Brosch R. Parkhill J. Garnier T. Churcher C. Harris D. Gordon S.V. Eiglmeier K. Gas S. Barry III, C.E. Tekaia F. Badcock K. Basham D. Brown D. Chillingworth T. Connor R. Davies R. Devlin K. Feltwell T. Gentles S. 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 R. Squares S. Sulston J.E. Taylor K. Whitehead S. Barrell B.G. Nature. 1998; 393: 537-544Crossref PubMed Scopus (6504) Google Scholar, 7Aoyama Y. Horiuchi T. Gotoh O. Noshiro M. Yoshida Y. J. Biochem. (Tokyo). 1998; 124: 694-696Crossref PubMed Scopus (71) Google Scholar). This raised the possibility that Mtb P450s could be novel drug targets, since fungal CYP51s are validated targets for azole drugs (e.g. fluconazole and clotrimazole) that coordinate the P450 heme iron and prevent oxidative transformation of lanosterol to ergosterol, with severe effects on fungal membrane integrity (8Odds F.C. Brown A.J. Brown A.J. Trends Microbiol. 2003; 11: 272-279Abstract Full Text Full Text PDF PubMed Scopus (873) Google Scholar). It was shown subsequently that several imidazole and triazole-based antifungals were effective anti-mycobacterial agents, with MIC values for various azoles being on the order of 0.1?20 ?g/ml for M. smegmatis (9Guardiola-Diaz H.M. Foster L.A. Mushrush D. Vaz A.D. Biochem. Pharmacol. 2001; 61: 1463-1470Crossref PubMed Scopus (111) Google Scholar, 10Jackson C.J. Lamb D.C. Kelly D.E. Kelly S.L. FEMS Microbiol. Lett. 2000; 192: 159-162Crossref PubMed Google Scholar, 11McLean K.J. Marshall K.R. Richmond A. Hunter I.S. Fowler K. Kieser T. Gurcha S.S. Besra G.S. Munro A.W. Microbiology. 2002; 148: 2937-2949Crossref PubMed Scopus (163) Google Scholar). However, CYP51B1 is not an essential gene for Mtb viability in culture and is unlikely to be a Mtb azole drug target (12Sassetti C.M. Boyd D.H. Rubin E.J. Mol. Microbiol. 2003; 48: 77-84Crossref PubMed Scopus (1998) Google Scholar). Instead, the Mtb CYP121 P450 was shown to bind very tightly to a range of azole antifungal drugs. Hitherto, there has been no evidence presented for the essentiality (or otherwise) of the CYP121 gene (Rv2276) in Mtb H37Rv (12Sassetti C.M. Boyd D.H. Rubin E.J. Mol. Microbiol. 2003; 48: 77-84Crossref PubMed Scopus (1998) Google Scholar). Although Mtb genome-wide transposon mutagenesis revealed that several P450 genes were not essential for viability or optimal growth in vitro, these studies did show that CYP128 was essential (12Sassetti C.M. Boyd D.H. Rubin E.J. Mol. Microbiol. 2003; 48: 77-84Crossref PubMed Scopus (1998) Google Scholar, 13Sassetti C.M. Boyd D.H. Rubin E.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12712-12717Crossref PubMed Scopus (486) Google Scholar). However, no data were presented regarding essentiality of CYP121 in these studies (12Sassetti C.M. Boyd D.H. Rubin E.J. Mol. Microbiol. 2003; 48: 77-84Crossref PubMed Scopus (1998) Google Scholar, 13Sassetti C.M. Boyd D.H. Rubin E.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12712-12717Crossref PubMed Scopus (486) Google Scholar) (see the Tuberculosis Animal Research and Gene Evaluation Taskforce (TARGET) site on the World Wide Web). Atomic structures of Mtb CYP51B1 and CYP121 were determined, both in complex with fluconazole and in ligand-free forms (15Podust L.M. Poulos T.L. Waterman M.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3068-3073Crossref PubMed Scopus (479) Google Scholar, 16Podust L.M. Yermalitskaya L.V. Lepesheva G.I. Podust V.N. Dalmasso E.A. Waterman M.R. Structure. 2004; 12: 1937-1945Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 17Seward H.E. Roujeinikova A. McLean K.J. Munro A.W. Leys D. J. Biol. Chem. 2006; 281: 39437-39443Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 18Leys D. Mowat C.G. McLean K.J. Richmond A. Chapman S.K. Walkinshaw M.D. Munro A.W. J. Biol. Chem. 2003; 278: 5141-5147Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). The CYP51B1-fluconazole complex structure demonstrated direct coordination of P450 heme iron by a fluconazole triazole nitrogen (15Podust L.M. Poulos T.L. Waterman M.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3068-3073Crossref PubMed Scopus (479) Google Scholar). The ligand-free structure of CYP121 revealed a highly constrained active site, and major reorganization of CYP121 structure was predicted to be necessary to facilitate direct azole coordination of the P450 heme iron (18Leys D. Mowat C.G. McLean K.J. Richmond A. Chapman S.K. Walkinshaw M.D. Munro A.W. J. Biol. Chem. 2003; 278: 5141-5147Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). However, the fluconazole-bound CYP121 structure showed a relative lack of structural perturbation and instead revealed an unusual mode of inhibitory coordination, with the azole nitrogen bridging to the heme iron via an interstitial water molecule (17Seward H.E. Roujeinikova A. McLean K.J. Munro A.W. Leys D. J. Biol. Chem. 2006; 281: 39437-39443Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). This type of binding was observed previously by Poulos and Howard (19Poulos T.L. Howard A.J. Biochemistry. 1987; 26: 8165-8174Crossref PubMed Scopus (224) Google Scholar) in studies of 2-phenylimidazole binding to P450cam. Recently, the structure of Mtb CYP130 (encoded by the Rv1256c gene absent from the genome of the Mycobacterium bovis BCG vaccine strain) was solved in complex with econazole, an effective antitubercular drug (20Ouellet H. Podust L.M. Ortiz de Montellano P.R. J. Biol. Chem. 2008; 283: 5069-5080Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 21Ahmad Z. Sharma S. Khuller G.K. Singh P. Faujdar J. Katoch V.M. Int. J. Antimicrob. Agents. 2006; 28: 543-544Crossref PubMed Scopus (51) Google Scholar). Econazole binds tightly to CYP130 (Kd = 1.93 ?m) (22McLean K.J. Cheesman M.R. Rivers S.L. Richmond A. Leys D. Chapman S.K. Reid G.A. Price N.C. Kelly S.M. Clarkson J. Smith W.E. Munro A.W. J. Inorg. Biochem. 2002; 91: 527-541Crossref PubMed Scopus (85) Google Scholar), although the Kd is higher than that for CYP121 (0.024 ?m; this study). However, CYP130 is a nonessential gene in Mtb strain CDC1551 (23Lamichhane G. Zignol M. Blades N.J. Geiman D.E. Dougherty A. Grosset J. Broman K.W. Bishai W.R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7213-7218Crossref PubMed Scopus (300) Google Scholar). The ligand-free CYP121 structure was the highest resolution P450 atomic structure solved to date (1.06 A?) and also revealed novel aspects of P450 structure, including (i) a distorted heme macrocycle caused by the displacement of a pyrrole group by a proline side chain, (ii) heme bound in two distinct conformations related by a 180? flip, and (iii) a relatively rigid active site hydrogen-bonded network of residues above the heme plane that defined the active site geometry and might contribute to a proton relay pathway to the heme iron (18Leys D. Mowat C.G. McLean K.J. Richmond A. Chapman S.K. Walkinshaw M.D. Munro A.W. J. Biol. Chem. 2003; 278: 5141-5147Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). In this study, we validate the importance of CYP121 to Mtb H37Rv viability by establishing its essentiality through CYP121 gene knock-out. We investigate the active site structure and role of several residues in the vicinity of the heme through a systematic determination of CYP121 mutant atomic structures, allied to detailed spectroscopic, thermodynamic, and azole binding analysis of these active site variants. We also report the MIC values for inhibition of Mtb H37Rv growth by azole drugs and compare these with their affinity (Kd values) for CYP121 and active site mutants thereof. Our study defines a new gene (CYP121) essential for viability of Mtb H37Rv and details the structural and biophysical properties of key CYP121 active site mutants. These data reveal important determinants of heme iron coordination and heme conformational and thermodynamic properties of CYP121 and enable a more detailed understanding of roles of residues either unique to CYP121 or broadly conserved in the P450 superfamily. A deletion delivery vector was generated by amplifying the upstream and downstream regions of CYP121 using primer pairs EUF5 (AAG CTT GAG ACG ACT CTG CTC CCA AC) and EUR5 (GGT ACC GCA CAG TGC ATA CGA GGA GA), and EUF6 (GGT ACC GCG CTG TTG AAA AAG ATG C) and EUR6 (GCG GCC GCA ACA CCG TTC TGG CGA TTA C) and cloning into p2NIL (24Parish T. Stoker N. Microbiology. 2000; 146: 1969-1975Crossref PubMed Scopus (399) Google Scholar) to generate an unmarked in-frame deletion. Restriction sites used for cloning are underlined. The gene cassette from pGOAL19 (24Parish T. Stoker N. Microbiology. 2000; 146: 1969-1975Crossref PubMed Scopus (399) Google Scholar) was cloned in as a PacI fragment to generate the final delivery vector pTACK121. A complementing vector (pCOLE121) was constructed by amplifying the CYP121 gene using primers CYP121D1 (CCT TAA TTA ATC GTT GAA TTG CTA CCA CCA) and CYP121D2 (CCT TAA TTA AGG TGC AAG GTC GAA ATT GTT) (PacI sites underlined) and subcloned into pAPA3 (25Parish T. Stoker N. J. Bacteriol. 2000; 182: 5715-5720Crossref PubMed Scopus (67) Google Scholar) under the control of the Ag85a promoter. Attempts to construct the CYP121 deletion strain using a two-step homologous recombination method were made (24Parish T. Stoker N. Microbiology. 2000; 146: 1969-1975Crossref PubMed Scopus (399) Google Scholar). A single crossover strain was generated by electroporating M. tuberculosis with 5 ?g of UV-treated pTACK121, and recombinants were selected on 100 ?g/ml hygromycin, 20 ?g/ml kanamycin, and 50 ?g/ml X-gal (24Parish T. Stoker N. Microbiology. 2000; 146: 1969-1975Crossref PubMed Scopus (399) Google Scholar). A single strain was streaked out in the absence of any antibiotics to allow the second crossover to occur. Double crossovers were selected and screened for using 2% (w/v) sucrose and 50 ?g/ml X-gal; white colonies were patch-tested for kanamycin and hygromycin sensitivity to ensure that they had lost the plasmid. PCR was used to screen for the presence of the wild type (WT) or deletion allele using primers CYP121D1 and CYP121D2. To generate a merodiploid strain, pCOLE121 was electroporated into the single crossover strain, and recombinants were isolated on 10 ?g/ml gentamicin, 100 ?g/ml hygromycin, 20 ?g/ml kanamycin, and 50 ?g/ml X-gal. A single recombinant was streaked out without antibiotics to allow a second crossover to occur, and double crossovers were isolated as before, except that gentamicin was included at all stages. PCR was used to screen for the presence of the WT or deletion allele using primers CYP121D1 and CYP121D2. Southern blot analysis was used to confirm the genotype of strains. Azole susceptibility testing on M. tuberculosis H37Rv was done by radiometric measurements using the BACTEC 460 system (BD Biosciences). A standard protocol was followed, using a biosafety level 3 biocontainment facility (25Parish T. Stoker N. J. Bacteriol. 2000; 182: 5715-5720Crossref PubMed Scopus (67) Google Scholar). The BACTEC vials contained Middlebrook 7H12B medium (BD Biosciences) with 14C-labeled palmitic acid as a carbon source. Four different azole drugs were tested for bacterial growth inhibition (econazole, miconazole, ketoconazole, and clotrimazole). Concentrated stocks of the azoles were made in DMSO and stored at -70 ?C until use. A range of azole drug concentrations were tested from 2 to 64 ?g/ml. The principle of the BACTEC system is described in more detail in the supplemental material. Short term culture filtrate and total cell lysate of M. tuberculosis H37Rv proteins were prepared as previously described (26Rosenkrands I. Aagaard C. Weldingh K. Brock I. Dziegiel M.H. Singh M. Hoff S. Ravn P. Andersen P. Tuberculosis. 2008; 88: 335-343Crossref PubMed Scopus (29) Google Scholar). Antiserum 1426D used for identification of CYP121 was raised by immunizing rats with purified, recombinant CYP121 delivered in Montanide ISA720 adjuvant (Seppic, Paris, France). SDS-PAGE and Western blots were performed as described under reducing conditions. To reduce responses to potential E. coli contaminants 1426D serum was absorbed with an E. coli extract for Western blot experiments (26Rosenkrands I. Aagaard C. Weldingh K. Brock I. Dziegiel M.H. Singh M. Hoff S. Ravn P. Andersen P. Tuberculosis. 2008; 88: 335-343Crossref PubMed Scopus (29) Google Scholar). Mutagenesis of CYP121 was done using the Stratagene QuikChange? site-directed mutagenesis kit for mutants A233G, F338H, S237A, S279A, R386L, and P346L. Mutant clones were confirmed by DNA sequencing (MWG Biotech) using generic T7 and T7 terminator oligonucleotide primers, allowing verification of entire gene sequences. Full methods for mutant production are given in the supplemental material. Primers used for CYP121 mutant gene generation are detailed in Table S1. The Mtb Rv2276 gene encoding WT CYP121 protein was expressed from E. coli HMS174 (DE3)/pKM2b transformants (22McLean K.J. Cheesman M.R. Rivers S.L. Richmond A. Leys D. Chapman S.K. Reid G.A. Price N.C. Kelly S.M. Clarkson J. Smith W.E. Munro A.W. J. Inorg. Biochem. 2002; 91: 527-541Crossref PubMed Scopus (85) Google Scholar). CYP121 protein was prepared from 10-liter cultures, as described previously (22McLean K.J. Cheesman M.R. Rivers S.L. Richmond A. Leys D. Chapman S.K. Reid G.A. Price N.C. Kelly S.M. Clarkson J. Smith W.E. Munro A.W. J. Inorg. Biochem. 2002; 91: 527-541Crossref PubMed Scopus (85) Google Scholar). Mutant CYP121 proteins were prepared similarly. The purity of CYP121 proteins was assessed by spectral properties (ratio of Soret absorption, at 416.5 nm in most cases, to protein-specific absorption at 280 nm, with an A416.5/A280 ratio of >1.8 indicating pure protein), and by SDS-PAGE analysis of protein samples (on 12% denaturing gels). CYP121 concentration was determined from the Soret absorption of the ferric enzyme in its ligand-free low spin state using ?416.5 = 110 mm-1 cm-1, as described previously (27Mclean K.J. Warman A.J. Seward H.E. Marshall K.R. Girvan H.M. Cheesman M.R. Waterman M.R. Munro A.W. Biochemistry. 2006; 45: 8427-8443Crossref PubMed Scopus (71) Google Scholar). For CYP121 mutants that displayed some high spin heme iron, a NO complex was generated by brief bubbling of ferric enzyme with NO gas. This gave rise to a single Fe(III)NO species (rather than a mixture of high spin and low spin Fe(III) species), and the absorption at the new Soret peak (437 nm; ?437 = 102 mm-1 cm-1) was then used to determine mutant enzyme concentration with reference to WT and as detailed in our previous work (27Mclean K.J. Warman A.J. Seward H.E. Marshall K.R. Girvan H.M. Cheesman M.R. Waterman M.R. Munro A.W. Biochemistry. 2006; 45: 8427-8443Crossref PubMed Scopus (71) Google Scholar). All mutant CYP121 enzymes were crystallized under the same conditions as reported previously for WT CYP121 (18Leys D. Mowat C.G. McLean K.J. Richmond A. Chapman S.K. Walkinshaw M.D. Munro A.W. J. Biol. Chem. 2003; 278: 5141-5147Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Complete data sets were obtained on single flash-cooled crystals at 100 K at the European Synchrotron Radiation Facility ID14 stations. Data were analyzed and merged using MOSFLM and SCALA (28Leslie, A. G. W. (1992) Joint CCP4 + ESF-EAMCB Newsletter on Protein Crystallography, No. 26Google Scholar, 29Evans P.R. Acta Crystallogr. Sect. D. 2005; 62: 72-82Crossref PubMed Scopus (3745) Google Scholar). Structures were refined using REFMAC5 (30Dodson E.J. Acta Crystallogr. Sect. D. 1997; 53: 240-255Crossref PubMed Scopus (13854) Google Scholar), using the WT structure as the starting model. The solvent model was built automatically using ARP/wARP (31Morris R.J. Perrakis A. Lamzin V.S. Methods Enzymol. 2003; 374: 229-244Crossref PubMed Scopus (473) Google Scholar). The oxidation state of the CYP121 heme may be ferrous, by analogy with the observed effects of synchrotron radiation on other heme proteins (32Beitlich T. Ku?hnel K. Schulze-Briese C. Shoeman R.L. Schlichting I. J. Synchrotron Radiat. 2007; 14: 11-23Crossref PubMed Scopus (129) Google Scholar). For final data and refinement statistics, see Table S2. Optical titrations for determination of azole binding constants (Kd values) were done as previously described (22McLean K.J. Cheesman M.R. Rivers S.L. Richmond A. Leys D. Chapman S.K. Reid G.A. Price N.C. Kelly S.M. Clarkson J. Smith W.E. Munro A.W. J. Inorg. Biochem. 2002; 91: 527-541Crossref PubMed Scopus (85) Google Scholar). Pure WT CYP121 and mutants (typically 1?3 ?m) were suspended in Buffer A in a 1-cm path length quartz cuvette, and a spectrum for the ligand-free form was recorded (250?800 nm) at 25 ?C on a Cary UV-50 Bio scanning spectrophotometer (Varian, UK). Azole ligands (clotrimazole, econazole, fluconazole, miconazole, ketoconazole, voriconazole, 2-phenylimidazole (2-PIM), and 4-phenylimidazole (4-PIM)) were titrated from concentrated stocks in DMSO solvent (apart from the phenylimidazoles, which were prepared in 60% ethanol) until no further optical perturbation was observed. Full information on titration methods is provided in the supplemental material. Induced optical change versus ligand concentration data were fitted using either a standard hyperbolic function (for data from 2-PIM and 4-PIM titrations) or to Equation 1, which provides a more accurate description of the binding of the antifungal azoles to WT and mutant forms of CYP121 (22McLean K.J. Cheesman M.R. Rivers S.L. Richmond A. Leys D. Chapman S.K. Reid G.A. Price N.C. Kelly S.M. Clarkson J. Smith W.E. Munro A.W. J. Inorg. Biochem. 2002; 91: 527-541Crossref PubMed Scopus (85) Google Scholar, 27Mclean K.J. Warman A.J. Seward H.E. Marshall K.R. Girvan H.M. Cheesman M.R. Waterman M.R. Munro A.W. Biochemistry. 2006; 45: 8427-8443Crossref PubMed Scopus (71) Google Scholar). Data were fitted using Origin software (OriginLab, Northampton, MA). Aobs=(Amax/2Et)×(S+Et+Kd)-(((S+Et+Kd)2-(4×S×Et))0.5)(Eq. 1) In Equation 1, Aobs is the observed absorbance change at ligand concentration S, Amax is the absorbance change at ligand saturation, Et is the P450 concentration, and Kd is the dissociation constant for the P450-ligand complex. All other spectral measurements for enzyme quantification and for establishing features of CYP121 in various redox states and in complex with other ligands (i.e. CO and NO) were also performed using a Cary 50 UV-visible spectrophotometer, either aerobically or under anaerobic conditions in a glove box (Belle Technology, Portesham, UK) for ferrous enzymes. Redox potentials for WT and mutant CYP121 enzymes were determined by anaerobic spectroelectrochemical titration according to established methods and as detailed in our previous studies of CYP121 and other P450s (22McLean K.J. Cheesman M.R. Rivers S.L. Richmond A. Leys D. Chapman S.K. Reid G.A. Price N.C. Kelly S.M. Clarkson J. Smith W.E. Munro A.W. J. Inorg. Biochem. 2002; 91: 527-541Crossref PubMed Scopus (85) Google Scholar, 27Mclean K.J. Warman A.J. Seward H.E. Marshall K.R. Girvan H.M. Cheesman M.R. Waterman M.R. Munro A.W. Biochemistry. 2006; 45: 8427-8443Crossref PubMed Scopus (71) Google Scholar, 33Daff S.N. Chapman S.K. Turner K.L. Holt R.A. Govindaraj S. Poulos T.L. Munro A.W. Biochemistry. 1997; 36: 13816-13821Crossref PubMed Scopus (207) Google Scholar, 34Dutton P.L. Methods Enzymol. 1978; 54: 411-435Crossref PubMed Scopus (729) Google Scholar, 35Lawson R.J. Leys D. Sutcliffe M.J. Kemp C.A. Cheesman M.R. Smith S.J. Clarkson J. Smith W.E. Haq I. Perkins J.B. Munro A.W. Biochemistry. 2004; 43: 12410-12426Crossref PubMed Scopus (55) Google Scholar). Full details are given in the supplemental material. UV-visible Spectroscopy?All UV-visible absorption spectra for oxidized WT and mutant CYP121 enzymes were recorded on a Cary UV-50 spectrophotometer in Buffer A at 25 ?C, as were spectra for azole ligand complexes and for Fe(III)NO and Fe(II)CO complexes. Resonance Raman?Resonance Raman (RR) spectra were obtained using 15-milliwatt, 406.7-nm radiation at the sample, from a Coherent Innova 300 krypton ion laser, and acquired using a Renishaw micro-Raman system 1000 spectrophotometer. The sample in Buffer A was held in a capillary under the microscope at a concentration of ~50 ?m, and an extended scan was obtained from 200 to 1700 cm-1 (total exposure time of 30 s). Ligand-free and fluconazole (100 ?m)-bound CYP121 samples were analyzed. Data processing, curve fitting, and band assignment was done using GRAMS/32 software (Thermo Scientific). EPR Spectroscopy?EPR spectra for the ferric CYP121 WT and mutant enzymes were recorded on a Bruker ER-300D series electromagnet and microwave source interfaced with a Bruker EMX control unit and fitted with an ESR-9 liquid helium flow cryostat (Oxford Instruments) and a dual mode microwave cavity from Bruker (ER-4116DM). Spectra were recorded at 10 K with a microwave power of 2.08 milliwatts and a modulation amplitude of 10 gauss for the CYP121 enzymes. Oxidized CYP121 and mutant samples were prepared in Buffer A. In order to establish if active site mutations disrupted electron transfer from NADPH to the P450 heme iron, kinetics of electron transport were analyzed using a Mtb H37Rv class I P450 redox system comprising the NAD(P)H-dependent flavoprotein reductase (FprA, encoded by Rv3106), the 3Fe-4S ferredoxin Fdx (encoded by Rv0763c adjacent to the CYP51B1 gene on the genome), and the relevant WT or mutant CYP121 protein. Conditions were 4 ?m CYP121 protein, 18 ?m Fdx, and 4 ?m FprA in 1 ml of Buffer A at 25 ?C. The buffer was deaerated and saturated with CO by extensive bubbling with the gas in a sealed quartz cuvette. Thereafter, the protein components were added (less than 20 ?l of total additions), and the reaction was initiated by injection of 300 ?m NADPH. Spectra (250?800 nm) were recorded regularly until no further change was detected and the P450 Fe(II)CO complexes were fully formed. The kinetics of complex formation were determined by plotting extent of Fe(II)CO complex formation against reaction time and fitting the resultant data using an exponential function and Origin software. Bacterial growth media (Tryptone, yeast extract) were from Melford Laboratories (Ipswich, Suffolk, UK). The 1-kb DNA ladder was from Promega. Azole drugs were from MP Biomedicals Inc. All other reagents were from Sigma and were of the highest grade available. We used a two-step homologous recombination strategy to demonstrate that CYP121 (Rv2276) is essential. A nonreplicating delivery vector carrying an unmarked in-frame deletion was constructed and used in a two-step homologous recombination process to isolate deletion mutants in either the WT or merodiploid. In the WT background, 40 double crossovers were screened by PCR, and all had the WT genotype. In contrast, we were able to isolate double crossovers with the deletion in the merodiploid background. Of eight strains screened, all had the deletion allele, and none had the WT allele. The genotype of the strains was confirmed by Southern analysis using two different restriction enzymes (Fig. 1). In parallel studies, we established that the CYP121 protein was produced by Mtb using immunological methods. Anti-CYP
Referência(s)