FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2012151111> ?p ?o ?g. }

• W2012151111 endingPage "4445" @default.
• W2012151111 startingPage "4434" @default.
• W2012151111 abstract "Mycolic acids are major and specific components of the cell envelope of Mycobacteria that include Mycobacterium tuberculosis, the causative agent of tuberculosis. Their metabolism is the target of the most efficient antitubercular drug currently used in therapy, and the enzymes that are involved in the production of mycolic acids represent important targets for the development of new drugs effective against multidrug-resistant strains. Among these are the S-adenosylmethionine-dependent methyltransferases (SAM-MTs) that catalyze the introduction of key chemical modifications in defined positions of mycolic acids. Some of these subtle structural variations are known to be crucial for both the virulence of the tubercle bacillus and the permeability of the mycobacterial cell envelope. We report here the structural characterization of the enzyme Hma (MmaA4), a SAM-MT that is unique in catalyzing the introduction of a methyl branch together with an adjacent hydroxyl group essential for the formation of both keto- and methoxymycolates in M. tuberculosis. Despite the high propensity of Hma to proteolytic degradation, the enzyme was produced and crystallized, and its three-dimensional structure in the apoform and in complex with S-adenosylmethionine was solved to about 2 Å. Thestructuresshowtheimportantroleplayedbythemodificationsfound within mycolic acid SAM-MTs, especially theα2-α3 motif and the chemical environment of the active site. Essential information with respect to cofactor and substrate binding, selectivity and specificity, and about the mechanism of catalytic reaction were derived. Mycolic acids are major and specific components of the cell envelope of Mycobacteria that include Mycobacterium tuberculosis, the causative agent of tuberculosis. Their metabolism is the target of the most efficient antitubercular drug currently used in therapy, and the enzymes that are involved in the production of mycolic acids represent important targets for the development of new drugs effective against multidrug-resistant strains. Among these are the S-adenosylmethionine-dependent methyltransferases (SAM-MTs) that catalyze the introduction of key chemical modifications in defined positions of mycolic acids. Some of these subtle structural variations are known to be crucial for both the virulence of the tubercle bacillus and the permeability of the mycobacterial cell envelope. We report here the structural characterization of the enzyme Hma (MmaA4), a SAM-MT that is unique in catalyzing the introduction of a methyl branch together with an adjacent hydroxyl group essential for the formation of both keto- and methoxymycolates in M. tuberculosis. Despite the high propensity of Hma to proteolytic degradation, the enzyme was produced and crystallized, and its three-dimensional structure in the apoform and in complex with S-adenosylmethionine was solved to about 2 Å. Thestructuresshowtheimportantroleplayedbythemodificationsfound within mycolic acid SAM-MTs, especially theα2-α3 motif and the chemical environment of the active site. Essential information with respect to cofactor and substrate binding, selectivity and specificity, and about the mechanism of catalytic reaction were derived. Mycolic acids, α-branched β-hydroxylated long chain fatty acids, are the hallmark of the Mycobacterium genus that comprises the causative agents of human diseases such as tuberculosis and leprosy, Mycobacterium tuberculosis and Mycobacterium leprae, respectively. These major cell envelope components play an important role in the structure and function of the mycobacterial cell envelope (1.Daffé M. Draper P. Adv. Microb. Physiol. 1998; 39: 131-203Crossref PubMed Google Scholar, 2.Goren M.B. Brennan P.J. Youmans G.P. Tuberculosis. W. B. Saunders Co., Philadelphia, PA1979: 63-193Google Scholar). For instance, mycolic acids attached to the cell wall arabinogalactanareorganizedwithotherlipidstoformanouterpermeabilitybarrier with an extremely low fluidity that confers an exceptional low permeability to mycobacteria and may explain their intrinsic resistance to many antibiotics (3.Brennan P.J. Nikaido H. Annu. Rev. Biochem. 1995; 64: 29-63Crossref PubMed Scopus (1563) Google Scholar). Similarly, trehalose mycolates have been implicated in numerous biological functions related both to the physiology and virulence of M. tuberculosis (1.Daffé M. Draper P. Adv. Microb. Physiol. 1998; 39: 131-203Crossref PubMed Google Scholar). Numerous studies have been and are currently devoted to understanding the structures and biosynthesis of mycolic acids, primarily because they are specific to the Mycobacterium genus, and their metabolism is the only clearly identified target inhibited by the major anti-tubercular drug isoniazid (4.Winder F.G. Collins P.B. J. Gen. Microbiol. 1970; 63: 41-48Crossref PubMed Scopus (174) Google Scholar, 5.Takayama K. Wang L. David H.L. Antimicrob. Agents Chemother. 1972; 2: 29-35Crossref PubMed Scopus (230) Google Scholar, 6.Davidson L.A. Takayama K. Antimicrob. Agents Chemother. 1979; 16: 104-105Crossref PubMed Scopus (49) Google Scholar, 7.Quémard A. Lacave C. Lanéelle G. Antimicrob. Agents Chemother. 1991; 35: 1035-1039Crossref PubMed Scopus (106) Google Scholar, 8.Rozwarski D.A. Grant G.A. Barton D.H. Jacobs Jr., W.R. Sacchettini J.C. Science. 1998; 279: 98-102Crossref PubMed Scopus (613) Google Scholar). With the resurgence of tuberculosis infections caused by multidrug-resistant strains and the need for the development of new anti-tubercular drugs, deciphering the biosynthesis pathway leading to mycolates still represents a major objective. Although much work remains to be done to complete their biosynthetic scheme, it is known that two mycobacterial fatty acid synthases participate in the formation of all types of mycolates. Fatty acid synthase I is necessary to produce C16,18 and C22-26 saturated fatty acids, which may be either directly incorporated into mycolates as the α-branch chain or used as substrates of the acyl carrier protein-dependent elongation system, fatty acid synthase-II, to produce the long meromycolic chain (for a recent review, see Ref. 9.Takayama K. Wang C. Besra G.S. Clin. Microbiol. Rev. 2005; 18: 81-101Crossref PubMed Scopus (506) Google Scholar). Mycolic acids usually occur in mycobacterial species as a mixture of various related molecules with different chemical groups at the so-called “proximal” and “distal” positions of their meromycolic chain (Fig. 1A). In members of the M. tuberculosis complex (M. tuberculosis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium microti, and Mycobacterium canetti), three types of mycolates are commonly encountered (10.Daffé M. Lanéelle M.A. Asselineau C. Levy-Frebault V. David H. Ann. Microbiol. (Paris). 1983; 134: 241-256Google Scholar, 11.Minnikin D.E. Minnikin S.M. Parlett J.H. Goodfellow M. Magnusson M. Arch. Microbiol. 1984; 139: 225-231Crossref PubMed Scopus (98) Google Scholar). The least polar type I α-mycolates are composed of C76-82 fatty acids (12.Laval F. Lanéelle M.A. Deon C. Monsarrat B. Daffé M. Anal. Chem. 2001; 73: 4537-4544Crossref PubMed Scopus (101) Google Scholar) and contain two cis cyclopropyl groups. The more polar type III and IV mycolates consist of C82-89 (12.Laval F. Lanéelle M.A. Deon C. Monsarrat B. Daffé M. Anal. Chem. 2001; 73: 4537-4544Crossref PubMed Scopus (101) Google Scholar) and contain a cis or trans (with a methyl group on the vicinal carbon atom) cyclopropyl group at the proximal position and a keto or methoxy group (with a methyl group on the vicinal carbon atom) at the distal position (Fig. 1A). These discrete structural variations in mycolates may be of crucial biological importance since it has been shown that mutations resulting in the loss of these chemical functions, and particularly the keto and methoxy groups, profoundly modify the permeability of the cell envelope to solutes and severely affect the virulence and pathogenicity of the mutant strains in experimental infections (13.Yuan Y. Zhu Y. Crane D.D. Barry III, C.E. Mol. Microbiol. 1998; 29: 1449-1458Crossref PubMed Scopus (145) Google Scholar, 14.Glickman M.S. Cox J.S. Jacobs Jr., W.R. Mol. Cell. 2000; 5: 717-727Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar, 15.Dubnau E. Chan J. Raynaud C. Mohan V.P. Lanéelle M.A. Yu K. Quémard A. Smith I. Daffé M. Mol. Microbiol. 2000; 36: 630-637Crossref PubMed Scopus (255) Google Scholar, 16.Rao V. Fujiwara N. Porcelli S.A. Glickman M.S. J. Exp. Med. 2005; 201: 535-543Crossref PubMed Scopus (198) Google Scholar). Accordingly, the enzymic systems that introduce the chemical modifications in the mycolic acid chain merit special attention. One of the eight genes that encode putative mycolic acid S-adenosylmethionine (SAM) 2The abbreviations used are: SAM, S-adenosylmethionine; SAM-MT, S-adenosylmethionine-dependent methyltransferase; MT, methyltransferase; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; SAH, S-adenosylhomocystein; CTAB, cetyltrimethylammonium bromide; DDDMAB, didecyldimethylammonium bromide; AcpM, acyl carrier protein from M. tuberculosis; MES, 4-morpholineethanesulfonic acid; r.m.s.d., root mean square deviation.-dependent methyltransferases (MTs) in M. tuberculosis (17.Cole 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. Barrell B.G. Nature. 1998; 393: 537-544Crossref PubMed Scopus (6522) Google Scholar), the mma4 (18.Yuan Y. Barry III, C.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12828-12833Crossref PubMed Scopus (131) Google Scholar) or hma (15.Dubnau E. Chan J. Raynaud C. Mohan V.P. Lanéelle M.A. Yu K. Quémard A. Smith I. Daffé M. Mol. Microbiol. 2000; 36: 630-637Crossref PubMed Scopus (255) Google Scholar) gene, has been shown to be necessary and sufficient for the synthesis of both keto- and methoxymycolic acids. Indeed, inactivation of the gene resulted in the suppression of the synthesis of both types of mycolic acid (12.Laval F. Lanéelle M.A. Deon C. Monsarrat B. Daffé M. Anal. Chem. 2001; 73: 4537-4544Crossref PubMed Scopus (101) Google Scholar, 15.Dubnau E. Chan J. Raynaud C. Mohan V.P. Lanéelle M.A. Yu K. Quémard A. Smith I. Daffé M. Mol. Microbiol. 2000; 36: 630-637Crossref PubMed Scopus (255) Google Scholar, 19.Dinadayala P. Laval F. Raynaud C. Lemassu A. Lanéelle M.A. Lanéelle G. Daffé M. J. Biol. Chem. 2003; 278: 7310-7319Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Consistently, transformation of Mycobacterium smegmatis with the hma gene resulted in the production of large amounts of hydroxymycolic acids with an adjacent methyl branch, structurally related to the ketomycolic acids of M. tuberculosis (20.Dubnau E. Lanéelle M.A. Soares S. Benichou A. Vaz T. Prome D. Prome J.C. Daffé M. Quémard A. Mol. Microbiol. 1997; 23: 313-322Crossref PubMed Scopus (61) Google Scholar). Trace amounts of these hydroxymycolic acids were also detected in mycobacterial species producing keto- and/or methoxymycolates, further supporting the hypothesis that hydroxymycolic acids can be the precursors of both keto- and methoxymycolic acids (21.Quémard A. Lanéelle M.A. Marrakchi H. Prome D. Dubnau E. Daffé M. Eur. J. Biochem. 1997; 250: 758-763Crossref PubMed Scopus (34) Google Scholar). Analysis of the mycolic acids elaborated by the hma mutant showed that the strain accumulates a new type of α-mycolate in which a distal double bond replaces a cyclopropyl ring (19.Dinadayala P. Laval F. Raynaud C. Lemassu A. Lanéelle M.A. Lanéelle G. Daffé M. J. Biol. Chem. 2003; 278: 7310-7319Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Thus, the Hma protein would transfer a methyl group from the SAM cofactor and, subsequently or simultaneously, a water molecule onto the double bound of ethylene substrates, leading to the formation of an hydroxylated product (15.Dubnau E. Chan J. Raynaud C. Mohan V.P. Lanéelle M.A. Yu K. Quémard A. Smith I. Daffé M. Mol. Microbiol. 2000; 36: 630-637Crossref PubMed Scopus (255) Google Scholar, 18.Yuan Y. Barry III, C.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12828-12833Crossref PubMed Scopus (131) Google Scholar, 20.Dubnau E. Lanéelle M.A. Soares S. Benichou A. Vaz T. Prome D. Prome J.C. Daffé M. Quémard A. Mol. Microbiol. 1997; 23: 313-322Crossref PubMed Scopus (61) Google Scholar, 21.Quémard A. Lanéelle M.A. Marrakchi H. Prome D. Dubnau E. Daffé M. Eur. J. Biochem. 1997; 250: 758-763Crossref PubMed Scopus (34) Google Scholar) (Fig. 1B). Other evidence also suggests that SAM-dependent methyltransferases would operate on full-length meromycolic derivatives (13.Yuan Y. Zhu Y. Crane D.D. Barry III, C.E. Mol. Microbiol. 1998; 29: 1449-1458Crossref PubMed Scopus (145) Google Scholar, 22.Qureshi N. Sathyamoorthy N. Takayama K. J. Bacteriol. 1984; 157: 46-52Crossref PubMed Google Scholar). To understand the mechanism of catalysis of this reaction and as a prerequisite for drug design, we have studied the Hma protein and solved its three-dimensional structure in the apo-form and in complex with SAM to 2.1 and 2.0 Å of resolution, respectively. Analysis of the Mycolic Acid Profile of Recombinant M. smegmatis Strains—The hma (mmaA4, Rv0642c) gene from M. tuberculosis H37Rv was amplified by PCR from genomic DNA and first cloned into the BamHI restriction site of the pQE30 plasmid (Qiagen), downstream the poly-His-coding region. This construction removed the first methionine residue from the sequence deduced from the gene and added 12 residues, including a non-cleavable His6-tag at the N terminus, leading to a protein that contains 312 amino acid residues. Using this construct as a template, the hma gene alone or together with the poly-His tag (h-hma) was amplified by PCR and cloned into the EcoRI and HindIII sites of the mycobacterial expression vector pMV261. The strain M. smegmatis mc2155 was transformed by electroporation (23.Stover C.K. de la Cruz V.F. Fuerst T.R. Burlein J.E. Benson L.A. Bennett L.T. Bansal G.P. Young J.F. Lee M.H. Hatfull G.F. Snapper S.B. Barletta R.G. Jacobs Jr., W.R. Bloom B.R. Nature. 1991; 351: 456-460Crossref PubMed Scopus (1209) Google Scholar) with the plasmid pMV261, pMV261::hma, or pMV261::h-hma. The recombinant strains were selected on 7H10 medium (Middlebrook) supplemented with 0.2% glycerol and 10 μg/ml kanamycin. Isolated colonies were used to inoculate cultures grown on the same medium. Bacteria were submitted to saponification as previously described (12.Laval F. Lanéelle M.A. Deon C. Monsarrat B. Daffé M. Anal. Chem. 2001; 73: 4537-4544Crossref PubMed Scopus (101) Google Scholar). After methylation using diazomethane, the mycolate patterns were analyzed by TLC on silica gel 60-coated plates (0.25 mm thickness, Macherey-Nagel) with elution in dichloromethane. Purified mycolic acid methyl esters were used as standards (10.Daffé M. Lanéelle M.A. Asselineau C. Levy-Frebault V. David H. Ann. Microbiol. (Paris). 1983; 134: 241-256Google Scholar). Fatty acid methyl esters were revealed by spraying molybdophosphoric acid (10% in ethanol) and charring. Expression and Purification of Hma for Structural Studies—The construction used for protein expression and purification was obtained by cloning the hma gene into the NdeI and BamHI sites of the expression vector pET-15b (Novagen). This construction removed the first three residues (Met-Thr-Arg) from the sequence deduced from the gene and added 20 residues, including a 17-residue-long cleavable His6-tag at the N terminus, leading to a fusion protein that contains 318 amino acid residues. The overexpression of the recombinant Hma fusion protein was carried out in Escherichia coli BL21(DE3)pLysS. Cultures were grown at 37 °C in Luria broth supplemented with 50 μg/ml ampicillin. One millimolar isopropyl-1-thio-β-d-galactopyranoside was added for induction when cell density reached an A600 of ∼0.5-0.9, and cell cultures were incubated for another 3 h at 37°C. Cells were harvested by centrifugation at 3000 × g for 10 min at 4 °C, and the pellets were washed with 50 mm MES-NaOH, 0.3 m NaCl, pH 6.5, and placed overnight at -20 °C. Bacteria were then lysed by sonication and centrifuged at 5000 × g for 1 h at 4 °C. Recombinant Hma was purified by using fast protein liquid chromatography on an ÄKTA Purifier system (Amersham Biosciences). First, the supernatant was applied to a chelating-Sepharose nickel affinity column (Amersham Biosciences). After extensive washes with 5 mm imidazole in 50 mm MES-NaOH, 0.3 m NaCl, pH 6.5, the protein was eluted with 0.15 m imidazole in the same buffer. Fractions corresponding to the protein were pooled and concentrated, and the engineered His6 tag was removed by thrombin (Novagen) (2 units/mg of protein, 4 h at 20 °C) if necessary. Concentrated tagged and untagged proteins were further purified by gel filtration with a HiLoad 16/60 Superdex-75 prepgrade chromatography column (Amersham Biosciences) and both eluted with 50 mm MES-NaOH, 0.15 m NaCl, 2 mm EDTA, 0.2 mm phenylmethylsulfonyl fluoride, pH 6.5. Fractions containing pure recombinant proteins were pooled, concentrated to 3-10 mg/ml using Centricon YM-10 (Millipore) devices, and stored at 4 °C. Protein concentration was determined by measuring UV spectra (A280 = 0.93 at 1 mg/ml, 1 cm). Electrophoretic Analysis and Mass Spectrometry—Protein purity was checked throughout purification using SDS-PAGE with a 15% acrylamide concentration. For mass spectrometry, SDS-PAGE separations were conducted using 12% polyacrylamide gels stained with Coomassie Brilliant Blue. Passive elution of proteins from polyacrylamide gels was achieved as described in Kurth and Stoffel (24.Kurth J. Stoffel W. Biol. Chem. Hoppe-Seyler. 1990; 371: 675-685Crossref PubMed Scopus (29) Google Scholar) and Claverol et al. (25.Claverol S. Burlet-Schiltz O. Gairin J.E. Monsarrat B. Mol. Cell. Proteomics. 2003; 2: 483-493Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Briefly, the gel pieces were excised and subsequently washed with H2O, and the proteins were allowed to diffuse out of the gel overnight at 37 °C by incubation in 20 μl of 0.1 m sodium acetate, 0.1% SDS, pH 8.2. Coomassie Brilliant Blue, SDS and salts were removed from the protein sample after passive elution by hydrophilic interaction chromatography using a ZipTipHPL according to the manufacturer's instructions (Millipore). Briefly, the ZipTipHPL was rehydrated in buffer A (H2O/CH3CN/CH3COOH 50/50/0.1, pH 5) and equilibrated with buffer B (H2O/CH3CN/CH3COOH 10/90/0.1, pH 5.5). Protein eluates were diluted in 200 μl of buffer B and loaded onto the ZipTipHPL. Salts were removed by washing with buffer B, and proteins were eluted with 4 μlof H2O/CH3CN/HCOOH (49/50/1). Electrospray ionization analysis was performed using an electrospray ionization quadrupole-time of flight mass spectrometer (QSTAR Pulsar, Applied Biosystems, Foster City, CA) operating in positive mode. A potential of 1-2 kV was applied to the precoated nanoelectrospray needles (New Objective, Picotips, Econotips) in the ion source. Instrument operation, data acquisition, and analysis were performed using Analyst® QS 1.0 software and Bioanalyst TM extensions. After extraction of the protein from the gel spot and desalting, acetonitrile was evaporated from the eluate at room temperature. Five microliters of trypsin solution (Promega) at 12.5 ng/μl in 12.5 mm NH4HCO3 were added, and the sample was incubated overnight at 37 °C. MALDI-TOF mass spectroscopy analyses were performed on a MALDI-TOF/TOF instrument (4700 Proteomics Analyzer; Applied Biosystems). A 0.5-μl volume of trypsin digest was applied on the MALDI target plate with 0.3 μl of matrix solution (α-cyano-4-hydroxycinnamic; 5 mg/ml in H2O/acetonitrile/TFA, 50/50/0.1). Mass spectra were acquired in automated positive reflector mode, from m/z 700 to 3500 and calibrated with external calibration. Peak lists from peptide mass mapping spectra were compared manually with the theoretical molecular masses of the trypsin peptides of Hma. Crystallization and X-ray Data Collection—Crystallization was performed at 12 °C by vapor equilibration using the hanging-drop method. Protein samples were concentrated to 3-10 mg/ml in the appropriate buffer (50 mm MES, 50 mm NaCl, pH 6.5). Drops were prepared by mixing equal volumes of protein and reservoir solutions; reservoir volumes of 500 μl were used. Basic, extension, and low ionic screens from Sigma were systematically used for initial screenings. X-ray diffraction quality crystals of Hma were obtained for the His6-tagged protein in the presence of 4-28% polyethylene glycol 3350, pH 5-9. All crystals were cryocooled in a stream of nitrogen gas at 100 K after a 3-min immersion in the crystallization solution supplemented with 20% (v/v) glycerol and stored in liquid nitrogen if necessary. For preparation of the binary complex with the cofactor, crystals were soaked in a solution containing both the cryoprotectant and 50 mm S-adenosylmethionine for 2-3 min. The various crystal forms were evaluated in-house at 285 and 100 K using a Rigaku RU300 rotating-anode source operating at 50 kV and 90 mA and a MarResearch Mar345dtb image-plate area detector. Diffraction data used for structure determination and refinement were collected at multistation beam-line ID14 of the European Synchrotron Radiation Facility (Grenoble, France). Data Processing and Phasing—All crystallographic calculations were performed using the CCP4 suite (26.Collaborative Computational Project Number 4Acta Crystallogr. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19770) Google Scholar) as implemented in the graphical user interface (27.Potterton E. Briggs P. Turkenburg M. Dodson E. Acta Crystallogr. D Biol. Crystallogr. 2003; 59: 1131-1137Crossref PubMed Scopus (1070) Google Scholar). X-ray diffraction data were processed using MOS-FLM (28.Leslie A.G.W. Helliwell J.R. Machin P.A. Papiz M.Z. Proceedings of the Daresbury Study Weekend: Computational Aspects of Protein Crystal Data Analysis. Science and Engineering Research Council, Daresbury Laboratory, Warrington, United Kingdom1987: 39-50Google Scholar) and scaled with SCALA (29.Evans P.R. Proceedings of the CCP4 Study Weekend: Data Collection and Processing. Science and Engineering Research Council, Daresbury Laboratory, Warrington, United Kingdom1993: 114-122Google Scholar). The structure of Hma in its apo-form was solved using molecular replacement with the program Phaser (30.Storoni L.C. McCoy A.J. Read R.J. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 432-438Crossref PubMed Scopus (1099) Google Scholar) and the structure of apoCmaA1 (31.Huang C.C. Smith C.V. Glickman M.S. Jacobs Jr., W.R. Sacchettini J.C. J. Biol. Chem. 2002; 277: 11559-11569Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar) (PDB entry code 1KP9). The search model was truncated as a polyalanine except for strictly conserved residues among the family of mycolic acid SAM-MTs from M. tuberculosis. The structure of Hma in complex with its cofactor was solved using the refined model of apoHma and molecular replacement with the program Phaser to compensate for variation along the unit cell c axis. Model Building and Crystallographic Refinement—Model building of apoHma was first carried out with ARP/wARP using the “warpNtrace” automated procedure (32.Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2564) Google Scholar). The initial map used for the calculation corresponded to the molecular replacement solution where several regions of the protein with different conformation were removed. All structures were then constructed manually in sigmaA-weighted electron density maps (33.Read R.J. Acta Crystallogr. A. 1986; 42: 140-149Crossref Scopus (2036) Google Scholar) using TURBO-FRODO. Restrained refinements of the structures were performed with the program REFMAC (34.Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. D. Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13870) Google Scholar) using a bulk solvent correction based on the Babinet principle and minimizing a maximum likelihood target function. Solvent molecules were automatically added as neutral oxygen atoms using wARP (35.Perrakis A. Sixma T.K. Wilson K.S. Lamzin V.S. Acta Crystallogr. D Biol. Crystallogr. 1997; 53: 448-455Crossref PubMed Scopus (480) Google Scholar). In the last stages of refinement, TLS parameters were refined using a single group for the whole molecule, which resulted in a similar improvement of the R and Rfree values. Production of the Figures—Fig. 3A was produced using TopDraw from the CCP4 graphical user interface (27.Potterton E. Briggs P. Turkenburg M. Dodson E. Acta Crystallogr. D Biol. Crystallogr. 2003; 59: 1131-1137Crossref PubMed Scopus (1070) Google Scholar). Figs. 3B, 5, and 6 were produced using BobScript (36.Esnouf R.M. J. Mol. Graph. 1997; 15: 133-138Google Scholar). Fig. 3B was rendered using RASTER3D (37.Merritt E.A. Murphy M.E.P. Acta Crystallogr. D Biol. Crystallogr. 1994; 50: 869-873Crossref PubMed Scopus (2857) Google Scholar). Fig. 4 was produced using ESPript (38.Gouet P. Courcelle E. Stuart D. Metoz F. Bioinformatics. 1998; 15: 305-308Crossref Scopus (2530) Google Scholar) with a sequence alignment edited manually and secondary structure assignment performed with STRIDE (39.Frishman D. Argos P. Proteins. 1995; 23: 566-579Crossref PubMed Scopus (2046) Google Scholar). Figs. 7, 8, 9 were produced using PyMol (58.DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific, San Carlos, CA2002Google Scholar).FIGURE 5Structural variation among mycolic acid SAM-MTs. Stereo view of the superimposed α-carbon traces of Hma (black), CmaA1 (cyan), MmaA2 (green), PcaA (magenta), and CmaA2 (orange). Apo structures are represented by dotted lines.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6Cofactor binding site and active site architecture of mycolic acid SAM-MTs. Stereo image of the chemical environment of the cofactor and of a cationic lipid as observed in the structures of Hma-SAM (protein, dark gray carbon atoms; SAM, orange carbon atoms) and CmaA1-SAH-CTAB (protein, light gray carbon atoms; SAH, CTAB, and bicarbonate, yellow carbon atoms). Except Val-12 and Tyr-16 of CmaA1, all numbers are for residues of Hma. Water molecules of Hma are represented in orange.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4Sequence alignment for M. tuberculosis mycolic acid SAM-MTs. Sequences were displayed from top to bottom by decreasing order of homology with respect to Hma (percentages of identity/similarity: MmaA3, 59/74; CmaA1, 56/68; MmaA2, 53/65; PcaA, 51/65; UmaA1, 49/66; MmaA1, 48/64; CmaA2, 47/62). Sequence similarities are highlighted in red, whereas sequence identities are shown as white letters on a red background. Secondary structure elements (arrows for β-strands and coils for helices) of Hma in complex with SAM and of CmaA1 in complex with SAH and CTAB are indicated at the top. Secondary structure elements that participate to the embellishment pattern are in green. Green circles, cysteine residues of Hma; purple stars, residues that make contact with the cofactor. Residues of Hma that might ultimately be eliminated upon proteolytic cleavage are underlined.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 7Molecular surface topography of the α2-α3 motif of mycolic acid SAM-MTs and of the recognition motif of AcpM. All molecular surfaces were displayed using the same scale. Side chain atoms of hydrophobic residues are in gold. Side chain nitrogen atoms of basic or acidic residues are in blue and red, respectively. The serine residue of AcpM that bears the 4′-phosphopantetheine prosthetic group is in green. The α2-α3 motif of SAM-MTs and the second and third helices of the AcpM bundle are displayed as ribbons.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 8Closed and open state of the mycolic acid SAM-MTs hydrophobic tunnel. Shown is a Close view inside the α2-α3 motif (vivid colors) toward the hydrophobic tunnel comparing the aperture size in Hma-SAM (left) and CmaA1-SAH-CTAB (right). CTAB is in pink, and CmaA1 atoms within 4 Å of the cationic detergent are in purple. The hydrophobic residues directly restricting the aperture were labeled. Dots represent van der Waals surfaces.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 9Structural variation in the α2-α3 motif of mycolic acid SAM-MTs. SAM and CTAB from the structures of Hma-SAM and CmaA1-SAH-CTAB, respectively, have been represented as sticks on the left. Selected dimensions are indicated.View Large Image Figure Vi" @default.
• W2012151111 created "2016-06-24" @default.
• W2012151111 creator A5005925862 @default.
• W2012151111 creator A5031332465 @default.
• W2012151111 creator A5035652134 @default.
• W2012151111 creator A5037775438 @default.
• W2012151111 creator A5041220789 @default.
• W2012151111 creator A5052662538 @default.
• W2012151111 creator A5069188437 @default.
• W2012151111 date "2006-02-01" @default.
• W2012151111 modified "2023-10-15" @default.
• W2012151111 title "Further Insight into S-Adenosylmethionine-dependent Methyltransferases" @default.
• W2012151111 cites W1504706957 @default.
• W2012151111 cites W1520730837 @default.
• W2012151111 cites W1552418306 @default.
• W2012151111 cites W1643004344 @default.
• W2012151111 cites W1971521753 @default.
• W2012151111 cites W1976558061 @default.
• W2012151111 cites W1985629904 @default.
• W2012151111 cites W1985649033 @default.
• W2012151111 cites W1986191025 @default.
• W2012151111 cites W1988255658 @default.
• W2012151111 cites W1993404146 @default.
• W2012151111 cites W1994342014 @default.
• W2012151111 cites W1996071493 @default.
• W2012151111 cites W2001641653 @default.
• W2012151111 cites W2009729452 @default.
• W2012151111 cites W2016765195 @default.
• W2012151111 cites W2017684948 @default.
• W2012151111 cites W2021805040 @default.
• W2012151111 cites W2023746875 @default.
• W2012151111 cites W2027340405 @default.
• W2012151111 cites W2031206118 @default.
• W2012151111 cites W2031782903 @default.
• W2012151111 cites W2034248706 @default.
• W2012151111 cites W2038840577 @default.
• W2012151111 cites W2047433615 @default.
• W2012151111 cites W2063259356 @default.
• W2012151111 cites W2065903108 @default.
• W2012151111 cites W2073632605 @default.
• W2012151111 cites W2078248419 @default.
• W2012151111 cites W2078290231 @default.
• W2012151111 cites W2078597516 @default.
• W2012151111 cites W2087375033 @default.
• W2012151111 cites W2090416400 @default.
• W2012151111 cites W2092026968 @default.
• W2012151111 cites W2092987196 @default.
• W2012151111 cites W2093712250 @default.
• W2012151111 cites W2095433835 @default.
• W2012151111 cites W2097443883 @default.
• W2012151111 cites W2099258581 @default.
• W2012151111 cites W2112021008 @default.
• W2012151111 cites W2139106269 @default.
• W2012151111 cites W2139360890 @default.
• W2012151111 cites W2140981202 @default.
• W2012151111 cites W2141920771 @default.
• W2012151111 cites W2150226488 @default.
• W2012151111 cites W2158023121 @default.
• W2012151111 cites W2164718888 @default.
• W2012151111 cites W2168428267 @default.
• W2012151111 cites W4238052567 @default.
• W2012151111 doi "https://doi.org/10.1074/jbc.m510250200" @default.
• W2012151111 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16356931" @default.
• W2012151111 hasPublicationYear "2006" @default.
• W2012151111 type Work @default.
• W2012151111 sameAs 2012151111 @default.
• W2012151111 citedByCount "65" @default.
• W2012151111 countsByYear W20121511112012 @default.
• W2012151111 countsByYear W20121511112013 @default.
• W2012151111 countsByYear W20121511112014 @default.
• W2012151111 countsByYear W20121511112015 @default.
• W2012151111 countsByYear W20121511112016 @default.
• W2012151111 countsByYear W20121511112017 @default.
• W2012151111 countsByYear W20121511112018 @default.
• W2012151111 countsByYear W20121511112019 @default.
• W2012151111 countsByYear W20121511112020 @default.
• W2012151111 countsByYear W20121511112021 @default.
• W2012151111 countsByYear W20121511112022 @default.
• W2012151111 countsByYear W20121511112023 @default.
• W2012151111 crossrefType "journal-article" @default.
• W2012151111 hasAuthorship W2012151111A5005925862 @default.
• W2012151111 hasAuthorship W2012151111A5031332465 @default.
• W2012151111 hasAuthorship W2012151111A5035652134 @default.
• W2012151111 hasAuthorship W2012151111A5037775438 @default.
• W2012151111 hasAuthorship W2012151111A5041220789 @default.
• W2012151111 hasAuthorship W2012151111A5052662538 @default.
• W2012151111 hasAuthorship W2012151111A5069188437 @default.
• W2012151111 hasBestOaLocation W20121511111 @default.
• W2012151111 hasConcept C185592680 @default.
• W2012151111 hasConcept C33288867 @default.
• W2012151111 hasConcept C552990157 @default.
• W2012151111 hasConcept C55493867 @default.
• W2012151111 hasConcept C86803240 @default.
• W2012151111 hasConcept C91965660 @default.
• W2012151111 hasConceptScore W2012151111C185592680 @default.
• W2012151111 hasConceptScore W2012151111C33288867 @default.
• W2012151111 hasConceptScore W2012151111C552990157 @default.
• W2012151111 hasConceptScore W2012151111C55493867 @default.

• next