Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique, Université Paul Sabatier (UMR5089), 205 route de Narbonne, 31077 Toulouse cedex, France1
Centre de Biochimie Structurale (INSERM U554 CNRS UMR5048 UM1), 29 rue de Navacelles, 34090 Montpellier cedex, France2
Author for correspondence: A. Quémard. Tel: +33 5 61 17 55 76. Fax: +33 5 61 17 59 94. e-mail: annaik{at}ipbs.fr
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ABSTRACT |
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Keywords: ß-ketoacyl reductase, quaternary structure, enzymic activity, structural model, fluorescence spectroscopy
Abbreviations: ACP, acyl carrier protein; ESI, electrospray ionization; FAS, fatty acid synthetase; H-MabA, His-tagged MabA; INH, isoniazid; MALDI-TOF, matrix-associated laser desorption ionization-time of flight; KAR, ß-ketoacyl-ACP reductase; PDB, Protein Data Bank; SDR, short-chain dehydrogenases/reductases
a Present address: St Jude Childrens Research Hospital, Dept of Biochemistry, 322 N Lauderdale, Memphis, TN 38105, USA.
b Hedia Marrakchi and Stéphanie Ducasse contributed equally to this work.
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INTRODUCTION |
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The FAS-II system has been isolated from a non-pathogenic mycobacterium, M. smegmatis (Odriozola et al., 1977 ). It is a complex of several monofunctional enzymes that catalyses the ACP-dependent elongation of palmitoyl-CoA (C16) into C18C30 saturated fatty acids, using malonyl-CoA as an elongation unit (Bloch, 1977
). The mycobacterial FAS-II is an unusual type II system in that it elongates medium-chain-length substrates, while the other known bacterial systems perform de novo biosynthesis (Bloch, 1977
). Only some of the proteins forming FAS-II have been positively identified, namely ACP (Bloch, 1977
, Kremer et al., 2001
), and more recently InhA, KasA and mtFabD (Marrakchi et al., 2000
; Kremer et al., 2000
, 2001
). The inhA gene is assumed to form an operon together with the upstream ORF, mabA (fabG1) (Banerjee et al., 1994
). The latter encodes a protein whose predicted amino acid sequence displays similarities with ß-ketoacyl-ACP reductases (KARs or FabG), and a total soluble protein extract of Escherichia coli expressing mabA had KAR activity (Banerjee et al., 1998
). Cloning, overexpression and purification were performed in order to obtain the pure MabA protein for both biochemical and biophysical characterizations. Here we report enzymological and structural analyses of MabA that represent a fundamental step towards the design of inhibitors. Specific structural features of MabA, interrelated with its substrate specificity, could allow the development of new antibiotics directed against mycobacteria.
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METHODS |
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Purification of MabA protein.
Induced recombinant bacteria were washed with cold 50 mM potassium phosphate buffer pH 7·8 (buffer A), and resuspended in 4 ml of the same buffer supplemented with 500 mM NaCl and 5 mM imidazole. Cells were broken by one freezing/thawing cycle at -70 °C, in the presence of protease inhibitors [aprotinin, soybean trypsin-inhibitor, TLCK (N-p-tosyl-lysine chloromethyl ketone), pepstatin, leupeptin and PMSF] and 0·5 mg lysozyme ml-1. Nucleic acids were removed by DNase I (5 µg ml-1) and RNase A (10 µg ml-1) treatments in the presence of MgCl2 (10 mM) at 4 °C for 15 min. After centrifugation at 44000 g for 15 min, the supernatant supplemented with 10% (v/v) glycerol was applied to a Ni-NTA Agarose (Qiagen) column (0·5 ml bed volume). After extensive washes by 5 mM then 50 mM imidazole in buffer A supplemented with 500 mM NaCl, MabA was eluted with 175 mM imidazole in buffer A supplemented with 500 mM NaCl. Fractions containing the His-tagged MabA (H-MabA) were identified by SDS-PAGE, pooled, dialysed twice against 50 vols 50% (v/v) glycerol in 50 mM potassium phosphate buffer, pH 7·2, and stored at -20 °C.
The InhA protein from M. tuberculosis was overproduced in E. coli and purified as previously described (Quémard et al., 1995 ).
Mass spectrometry (MS).
Electrospray ionization (ESI) analysis of MabA was performed using a TSQ 700 (Finnigan MAT) quadrupole mass spectrometer. The protein was dissolved in methanol/water/acetic acid (50:49·5:0·5, by vol.) and introduced by a syringe pump (Harvard) at a flow rate of 5 µl min-1 into the electrospray source (5 kV, 250 °C). Nitrogen (pressure 40 p.s.i., 276 kPa) was used as nebulizing gas. MALDI-TOF spectra (in the positive mode) were acquired on a Voyager-DE STR Biospectrometry workstation (PerSeptive Biosystems), equipped with a pulsed nitrogen laser emitting at 337 nm, as described by Laval et al. (2001) , using an extraction voltage of 20 kV and 2,5-dihydroxybenzoic acid solution [10 mg ml-1 in water/acetonitrile, 8:2 (v/v)] as matrix.
Analytical size-exclusion chromatography.
A prepacked Sephacryl S-100HR column (Amersham Pharmacia Biotech), monitored by a BioCAD SPRINT system (PerSeptive Biosystems), was equilibrated in 50 mM potassium phosphate buffer, pH 6·8, containing 100 mM NaCl. H-MabA (0·7 mg) and InhA (1·1 mg) were separately eluted through the column with the same buffer, at a flow rate of 0·5 ml min-1. The column was calibrated in the same elution conditions by applying, in two separate elutions, the following standard proteins (0·51 mg of each protein): alcohol dehydrogenase (150 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa) and RNase A (13·7 kDa). The molecular mass of H-MabA was estimated from the calibration curve of elution volumes versus molecular masses.
Analytical ultracentrifugation.
Analytical ultracentrifugation experiments were performed in a Beckman Optima XL-I analytical ultracentrifuge using an An-55 four-hole rotor with six-channel Epon charcoal-filled centrepieces, and absorbance optics. Prior to the measurements, the protein samples were eluted through a Fast desalting column HR 10/10 (Amersham Pharmacia Biotech) in 20 mM MES, pH 6·4, 300 mM LiSO4 and 1 mM tris-(carboxyethyl)phosphine hydrochloride. Sedimentation equilibrium experiments were carried out at 4 °C on sample volumes of 110 µl, at loading MabA concentrations of 26, 39 and 78 µM, and at rotor speeds of 4872, 7015 and 14637 g (respectively 7500, 9000 and 13000 r.p.m.). Data were collected at 280 nm and runs were continued until there was no significant difference in scans taken 2 h apart. Data were analysed with the XL-A/XL-I Data Analysis software version 4.0 (Beckman). The partial specific volume (0·7223 ml g-1) of the recombinant H-MabA protein and the solvent density (1·04 g ml-1), at the experimental temperature, were calculated with help of the program Sednterp (Sedimentation Interpretation Program, version 1.05).
Preparation of FAS-I and FAS-II systems or cell-wall extract, and Western blots.
Cell-wall extract, FAS-I and FAS-II systems were prepared from M. smegmatis mc2155 as previously described (Marrakchi et al., 2000 ). An aliquot of total protein extract was kept after sonication of bacteria, during preparation of FAS-I and FAS-II systems. Samples were separated by 12% or 15% (w/v) polyacrylamide SDS-PAGE, and proteins transferred onto a nitrocellulose membrane. Polyclonal rabbit antibodies against pure H-MabA protein were used for Western blot analyses. Rabbit preimmune serum was used for control experiments. Antigenantibody interactions were revealed by colorimetric reaction, using alkaline phosphatasemouse anti-rabbit IgG conjugates.
Synthesis of different chain length ß-ketoacyl-CoAs.
ß-Ketoacyl-CoAs possessing 820 carbon atoms were synthesized as described by Vagelos & Alberts (1960) , with some modifications. For solubility reasons, for the C16 and C20 derivatives the deprotection step of the ketone function was realized by a transcetalization reaction, by incubating the ethylenic ketal derivatives in anhydrous acetone and p-toluenesulphonic acid (10 mM) at 56 °C, for 36 h. The derivatization into acyl-CoAs was performed at pH 8·7, in 600 mM Tris/tetrahydrofuran (1:1, v/v). The compounds obtained at each synthesis step were purified and their structure characterized by electron-impact MS, IR and NMR spectroscopies. The final ß-ketoacyl-CoA products were purified by chromatography on a C18 Sep-Pak cartridge equilibrated with 20 mM NaH2PO4 and elution with a 080% methanol gradient in water, and their structures verified by ESI-MS in the negative mode.
Enzyme assays and steady-state kinetics.
Kinetic parameters were determined spectrophotometrically by following NADPH oxidation at 340 nm using a thermostatted Uvikon 923 spectrophotometer (Kontron Instruments). Standard reactions were performed in a quartz cuvette in a total volume of 1 ml, at 25 °C, in 100 mM sodium phosphate buffer, pH 7·0, in the presence of fixed concentrations of NADPH and ß-ketoacyl-CoA; after equilibration of the baseline, reactions were started by adding a defined amount of H-MabA enzyme, and measurements performed for 35 min. Comparison of the initial reaction rates obtained for the different chain length ß-ketoacyl-CoAs at a low substrate concentration was performed at 2 µM ß-ketoacyl-CoA, 100 µM NADPH and 18360 nM H-MabA (depending on the substrate used). The steady-state Km value for NADPH was determined by measuring initial velocities at various concentrations of coenzyme and at fixed concentrations of acetoacetyl-CoA (400 µM) and of enzyme (36 nM). The Km values for ß-ketoacyl-CoAs and kcat values were measured by varying the concentration of each ß-ketoacyl-CoA and at fixed concentrations of NADPH (100 µM) and of enzyme (18144 nM, depending on the substrate). Data were fitted to the MichaelisMenten equation by least-squares fits to a hyperbola using the program GraphPad Prism to calculate the kinetic parameters. For MALDI-TOF MS analyses, reactions were performed in a total volume of 1 ml, at 25 °C, in the presence of 300 µM NADPH, 200 µM acetoacetyl-CoA and 800 nM H-MabA, in 20 mM Tris buffer, pH 7·0, and stopped after completion (monitored by A340 measurement).
Sequence comparison and molecular modelling.
Protein sequence database searches were performed with PSI-BLAST version 2.0.5 (Altschul et al., 1997 ), with default parameters. Alignment refinement was subsequently performed using the program TITO (Labesse & Mornon, 1998
) and structures of related enzymes (see text). MabA secondary structures (
-helices, ß-strands) were assigned during TITO processing, and the secondary structures derived by homology were used as additional restraints in the following modelling step (except for the stretch 48-GSGAPKG-54 due to its sequence enriched in glycine). The three-dimensional model was built using the quaternary structure deduced from the crystal structure of PDB1EDO (40% identical) as a template in the program MODELLER 4.0 (Sali & Blundell, 1993
) and assessed using Verify-3D (Eisenberg et al., 1997
) and PROSA (Sippl, 1993
). These three-dimensional structures were visualized on a UNIX workstation using the program InsightII (MSI, San Diego, USA).
Fluorescence spectroscopy.
Steady-state emission spectra were recorded on an ISS K2 spectrofluorimeter through a 8 nm bandwidth monochromator and corrected for buffer emission. Time-resolved data were collected in the frequency domain using ISS acquisition electronics and analysed with the software Globals Unlimited (Beechem et al., 1991 ). The excitation light was at 300 nm from the frequency-tripled output of a pulse-picked Tsunami Ti-Sa laser pumped by a Millennia X dye laser (Spectra Physics). The reference lifetime compound was N-acetyltryptophanamide in water (3 ns). Measurements were carried out using 6 µM H-MabA in 100 mM sodium phosphate buffer, pH 7·0, and 7% (w/v) glycerol, in the presence or absence of a ligand (at saturating concentration of NADP+ and/or Km concentration of ß-ketoacyl-CoA). Fluorescence spectroscopy in the presence of NADPH was not performed as the coenzyme absorption spectrum overlaps the emission spectrum of the protein tryptophan residue.
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RESULTS |
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Structural analysis of the MabA protein
Through PSI-BLAST searches (Altschul et al., 1997 ) performed in non-redundant databases, MabA appeared highly conserved among mycobacteria (8184% identity in M. tuberculosis, M. avium and M. smegmatis), while identity scores with other KARs (16 sequences) from various organisms, bacteria or plants, ranged from 29 to 43% over the whole sequence. The search for related three-dimensional structures indicated extended similarities with the short-chain dehydrogenases/reductases (SDR) superfamily (Rafferty et al., 1995
; Jörnvall et al., 1995
), or the reductases/epimerases/dehydrogenases (RED) superfamily (Labesse et al., 1994
), to which InhA belongs, the closest structure being the KAR from Brassica napus (PDB1EDO), with 40% identity. Most residues critical for cofactor binding and catalysis are well conserved in MabA sequence (Fig. 5
). Furthermore, in agreement with the kinetic data, the ß2 strand displays a sequence motif (Fig. 5
) typical of the NADP(H)-specific enzymes (Labesse et al., 1994
).
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Thus, the fluorescence data correlate with the measured kinetic parameters since they both show a major difference between the C4 and the longer substrates (Fig. 7, Tables 1
and 2
).
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DISCUSSION |
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Both MabA and InhA have a large substrate-binding pocket, adequate to accommodate the long acyl chains elongated by FAS-II. The substrate specificity of MabA is also consistent with that of the M. tuberculosis KasIII (or mtFabH) protein, believed to play the role of pivotal link between FAS-I and FAS-II systems (Choi et al., 2000 ). This specificity might partly reside in the relatively strong hydrophobicity of the MabA substrate-binding pocket, particularly in the bottom part, which appears globally more hydrophobic than that of the other known KARs. It is noteworthy that, in the crystal structure of the ternary complex InhANAD+C16 substrate, numerous hydrophobic residues interact with the long aliphatic chain of the substrate (Rozwarski et al., 1999
). The steric hindrance and local conformational changes induced by a long acyl chain interacting with residues lining the MabA substrate-binding pocket may explain the difference in fluorescence behaviour between the C4 and C8C16 substrates. Likewise, local rearrangements of the substrate-binding pocket of InhA and ThnR were observed upon binding of the substrate or an inhibitor (Rozwarski et al., 1999
; Andersson et al., 1996
).
The outbreak of resistant M. tuberculosis strains over the last 15 years has focused international attention on the need for new drug development. The structural and functional features of the MabA protein described here, especially its affinity for long-chain substrates, should allow the development of MabA-specific substrate analogues, as exemplified by thymidine monophosphate kinase of M. tuberculosis (Munier-Lehmann et al., 2001 ), which might lead to the design of novel antituberculous drugs.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Andersson, A., Jordan, D., Schneider, G. & Lindqvist, Y. (1996). Crystal structure of the ternary complex of 1,3,8-trihydroxynaphthalene reductase from Magnaporthe grisea with NADPH and an active-site inhibitor. Structure 4, 1161-1170.[Medline]
Banerjee, A., Dubnau, E., Quémard, A., Balasubramanian, V., Um, K. S., Wilson, T., Collins, D., de Lisle, G. & Jacobs, W. R.Jr (1994). inhA, a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis. Science 263, 227-230.[Medline]
Banerjee, A., Sugantino, M., Sacchettini, J. C. & Jacobs, W. R.Jr (1998). The mabA gene from the inhA operon of Mycobacterium tuberculosis encodes a 3-ketoacyl reductase that fails to confer isoniazid resistance. Microbiology 144, 2697-2704.[Abstract]
Beechem, J. M., Gratton, E., Ameloot, M. A., Knutson, J. R. & Brand, L. (1991). The global analysis of fluorescence intensity and anisotropy decay data: second-generation theory and programs. In Fluorescence Spectroscopy , pp. 241-301. Edited by J. R. Lakowicz. New York:Plenum.
Bloch, K. (1977). Control mechanisms for fatty acid synthesis in Mycobacterium smegmatis. Adv Enzymol 45, 1-84.[Medline]
Choi, K. H., Kremer, L., Besra, G. S. & Rock, C. O. (2000). Identification and substrate specificity of beta-ketoacyl (acyl carrier protein) synthase III (mtFabH) from Mycobacterium tuberculosis. J Biol Chem 275, 28201-28207.
Daffé, M. & Draper, P. (1998). The envelope layers of mycobacteria with reference to their pathogenicity. Adv Microb Physiol 39, 131-203.[Medline]
Eisenberg, D., Luthy, R. & Bowie, J. U. (1997). VERIFY3D: assessment of protein models with three-dimensional profiles. Methods Enzymol 277, 396-404.[Medline]
Fisher, M., Kroon, J. T. M., Martindale, W., Stuitje, A. R., Slabas, A. R. & Rafferty, J. B. (2000). The X-ray structure of Brassica napus beta-keto acyl carrier protein reductase and its implications for substrate binding and catalysis. Structure 8, 339-347.[Medline]
Gouet, P., Courcelle, E., Stuart, D. I. & Metoz, F. (1999). ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics 15, 305-308.
Jörnvall, H., Persson, B., Krook, M., Atrian, S., Gonzalez-Duarte, R., Jeffery, J. & Ghosh, D. (1995). Short-chain dehydrogenases/reductases (SDR). Biochemistry 34, 6003-6013.[Medline]
Kraulis, P. J. (1991). MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J Appl Cryst 24, 946-950.
Kremer, L., Douglas, J. D., Baulard, A. R. & 9 other authors (2000). Thiolactomycin and related analogues as novel anti-mycobacterial agents targeting KasA and KasB condensing enzymes in Mycobacterium tuberculosis. J Biol Chem 275, 1685716864.
Kremer, L., Nampoothiri, K. M., Lesjean & 7 other authors (2001). Biochemical characterization of acyl carrier protein (AcpM) and malonyl-CoA:AcpM transacylase (mtFabD), two major components of Mycobacterium tuberculosis fatty acid synthase II. J Biol Chem 276, 2796727974.
Labesse, G. & Mornon, J. P. (1998). A Tool for Incremental Threading Optimization (TITO) to help alignment and modelling of remote homologs. Bioinformatics 14, 206-211.[Abstract]
Labesse, G., Vidal-Cros, A., Chomilier, J., Gaudry, M. & Mornon, J.-P. (1994). Structural comparisons lead to the definition of a new superfamily of NAD(P)(H)-accepting oxidoreductases: the single-domain reductases/epimerases/dehydrogenases (the RED family). Biochem J 304, 95-99.[Medline]
Lakowicz, J. R. (1983). Protein fluorescence. In Principles of Fluorescence Spectroscopy , pp. 342-381. Edited by J. R. Lakowicz. New York:Plenum.
Laval, F., Lanéelle, M. A., Déon, C., Montsarrat, B. & Daffé, M. (2001). Accurate molecular mass determination of mycolic acids by MALDI-TOF mass spectrometry. Anal Chem 73, 4537-4544.[Medline]
Liu, J., Barry, C. E.3rd, Besra, G. S. & Nikaido, H. (1996). Mycolic acid structure determines the fluidity of the mycobacterial cell wall. J Biol Chem 271, 29545-29551.
Marrakchi, H., Lanéelle, G. & Quémard, A. (2000). InhA, a target of the antituberculous drug isoniazid, is involved in a mycobacterial fatty acid elongation system, FAS-II. Microbiology 146, 289-296.
Mdluli, K., Slayden, R. A., Zhu, Y., Ramaswamy, S., Pan, X., Mead, D., Crane, D. D., Musser, J. M. & Barry, C. E.3rd (1998). Inhibition of a Mycobacterium tuberculosis beta-ketoacyl ACP synthase by isoniazid. Science 280, 1607-1610.
Munier-Lehmann, H., Chaffotte, A., Pochet, S. & Labesse, G. (2001). Thymidylate kinase of Mycobacterium tuberculosis: a chimera sharing properties common to eukaryotic and bacterial enzymes. Protein Sci 10, 1195-1205.
Nakajima, K., Kato, H., Oda, J., Yamada, Y. & Hashimoto, T. (1999). Site-directed mutagenesis of putative substrate-binding residues reveals a mechanism controlling the different stereospecificities of two tropinone reductases. J Biol Chem 274, 16563-16568.
Odriozola, J. M., Ramos, J. A. & Bloch, K. (1977). Fatty acid synthetase activity in Mycobacterium smegmatis. Characterization of the acyl carrier protein-dependent elongating system. Biochim Biophys Acta 488, 207-217.[Medline]
Quémard, A., Lacave, C. & Lanéelle, G. (1991). Isoniazid inhibition of mycolic acid synthesis by cell free extracts of sensitive and resistant strains of Mycobacterium aurum. Antimicrob Agents Chemother 35, 1035-1039.[Medline]
Quémard, A., Sacchettini, J. C., Dessen, A., Vilchèze, C., Bittman, R., Jacobs, W. R.Jr & Blanchard, J. S. (1995). Enzymatic characterization of the target for isoniazid in Mycobacterium tuberculosis. Biochemistry 34, 8235-8241.[Medline]
Quémard, A., Dessen, A., Sugantino, M., Jacobs, W. R.Jr, Sacchettini, J. C. & Blanchard, J. S. (1996). Binding of catalase-peroxidase-activated isoniazid to wild-type and mutant Mycobacterium tuberculosis enoyl-ACP reductases. J Am Chem Soc 118, 1561-1562.
Rafferty, J. B., Simon, J. W., Baldock, C., Artymiuk, P. J., Baker, P. J., Stuitje, A. R., Slabas, A. R. & Rice, D. W. (1995). Common themes in redox chemistry emerge from the X-ray structure of oilseed rape (Brassica napus) enoyl acyl carrier protein reductase. Structure 3, 927-938.[Medline]
Rozwarski, D. A., Grant, G. A., Barton, D. H. R., Jacobs, W. R.Jr & Sacchettini, J. C. (1998). Modification of the NADH of the isoniazid target (InhA) from Mycobacterium tuberculosis. Science 279, 98-102.
Rozwarski, D. A., Vilchèze, C., Sugantino, M., Bittman, R. & Sacchettini, J. C. (1999). Crystal structure of the Mycobacterium tuberculosis enoyl-ACP reductase, InhA, in complex with NAD+ and a C16 fatty acyl substrate. J Biol Chem 274, 15582-15589.
Sali, A. & Blundell, T. L. (1993). Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 234, 779-815.[Medline]
Sheldon, P. S., Kekwick, R. G. O., Sidebottom, C., Smith, C. G. & Slabas, A. R. (1990). 3-Oxoacyl-(acyl-carrier protein) reductase from avocado (Persea americana) fruit mesocarp. Biochem J 271, 713-720.[Medline]
Sheldon, P. S., Kekwick, R. G., Smith, C. G., Sidebottom, C. & Slabas, A. R. (1992). 3-Oxoacyl-[ACP] reductase from oilseed rape (Brassica napus). Biochim Biophys Acta 1120, 151-159.[Medline]
Shimakata, T. & Stumpf, P. K. (1982). Purification and characterization of ß-ketoacyl-[acyl-carrier-protein] reductase, of ß-hydroxyacyl-[acyl-carrier-protein] dehydrase, and enoyl-[acyl-carrier-protein] reductase from Spinacea oleracea leaves. Arch Biochem Biophys 218, 77-91.[Medline]
Sippl, M. J. (1993). Recognition of errors in three-dimensional structures of proteins. Proteins 17, 355-362.[Medline]
Takayama, K., Wang, L. & David, H. L. (1972). Effect of isoniazid on the in vivo mycolic acid synthesis, cell growth, and viability of Mycobacterium tuberculosis. Antimicrob Agents Chemother 2, 29-35.[Medline]
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673-4680.[Abstract]
Thompson, J. E., Basarab, G. S., Andersson, A., Lindqvist, Y. & Jordan, D. B. (1997). Trihydroxynaphthalene reductase from Magnaporthe grisea: realization of an active center inhibitor and elucidation of the kinetic mechanism. Biochemistry 36, 1852-1860.[Medline]
Vagelos, P. R. & Alberts, A. W. (1960). Chemical synthesis of ß-ketooctanoyl coenzyme A. Anal Biochem 1, 8-16.[Medline]
Vilchèze, C., Morbidoni, H. R., Weisbrod, T. R., Iwamoto, H., Sacchettini, J. C. & Jacobs, W. R.Jr (2000). Inactivation of the inhA-encoded fatty acid synthase II (FASII) enoyl-acyl carrier protein reductase induces accumulation of the FASI end products and cell lysis of Mycobacterium smegmatis. J Bacteriol 182, 4059-4067.
Zabinski, R. F. & Blanchard, J. S. (1997). The requirement for manganese and oxygen in the isoniazid-dependent inactivation of Mycobacterium tuberculosis enoyl reductase. J Am Chem Soc 119, 2331-2332.
Received 9 November 2001;
revised 28 November 2001;
accepted 29 November 2001.