cDNA cloning and expression of Brassica napus enoyl-acyl carrier protein reductase in Escherichia coli

The onset of storage lipid biosynthesis during seed development in the oilseed crop Brassica napus (rape seed) coincides with a drastic qualitative and quantitative change in fatty acid composition. During this phase of storage lipid biosynthesis, the enzyme activities of the individual components of the fatty acid synthase system increase rapidly. We describe a rapid and simple purification procedure for the plastidlocalized NADH-dependent enoyl-acyl carrier protein reductase from developing B. napus seed, based on its affinity towards the acyl carrier protein (ACP). The purified protein was N-terminally sequenced and used to raise a potent antibody preparation. Immuno-screening of a seed-specific gt11 cDNA expression library resulted in the isolation of enoyl-ACP reductase cDNA clones. DNA sequence analysis of an apparently full-length cDNA clone revealed that the enoyl-ACP reductase mRNA is translated into a precursor protein with a putative 73 amino acid leader sequence which is removed during the translocation of the protein through the plastid membrane. Expression studies in Escherichia coli demonstrated that the full-length cDNA clone encodes the authentic B. napus NADH-dependent enoyl-ACP reductase. Characterization of the enoyl-ACP reductase genes by Southern blotting shows that the allo-tetraploid B. napus contains two pairs of related enoyl-ACP reductase genes derived from the two distinct genes found in both its ancestors, Brassica oleracea and B. campestris. Northern blot analysis of enoyl-ACP reductase mRNA steady-state levels during seed development suggests that the increase in enzyme activity during the phase of storage lipid accumulation is regulated at the level of gene expression.     

The use of a hybrid genetic system to study the functional relationship between prokaryotic and plant multi-enzyme fatty acid synthetase complexes

Fatty acid synthesis in bacteria and plants is catalysed by a multi-enzyme fatty acid synthetase complex (FAS II) which consists of separate monofunctional polypeptides. Here we present a comparative molecular genetic and biochemical study of the enoyl-ACP reductase FAS components of plant and bacterial origin. The putative bacterial enoyl-ACP reductase gene (envM) was identified on the basis of amino acid sequence similarities with the recently cloned plant enoyl-ACP reductase. Subsequently, it was unambiguously demonstrated by overexpression studies that theenvM gene encodes the bacterial enoyl-ACP reductase. An anti-bacterial agent called diazaborine was shown to be a specific inhibitor of the bacterial enoyl-ACP reductase, whereas the plant enzyme was insensitive to this synthetic antibiotic. The close functional relationship between the plant and bacterial enoyl-ACP reductases was inferred from genetic complementation of anenvM mutant ofEscherichia coli. Ultimately,envM gene-replacement studies, facilitated by the use of diazaborine, demonstrated for the first time that a single component of the plant FAS system can functionally replace its counterpart within the bacterial multienzyme complex. Finally, lipid analysis of recombinantE. coli strains with the hybrid FAS system unexpectedly revealed that enoyl-ACP reductase catalyses a rate-limiting step in the elongation of unsaturated fatty acids.     

Studies on the multi-enzyme complex of yeast fatty-acid synthetase. Reversible dissociation and isolation of two polypeptide chains.

1. The multi-enzyme complex of fatty acid synthetase, Mr 2300,000, was dissociated by acylation with dimethyl maleic anhydride under conditions which lead to an acylation of about 30% of the epsilon amino groups of lysine. The complete dissociation into the subunits alpha and beta is demonstrated by analytical ultracentrifugation as well as disc gel electrophoresis. 2. This dissociation is reversible. Hydrolysis of the resulting protein dicarboxylic acid monoamides under mildly acidic conditions leads to the unmodified subunits, which can be reconstituted to form a complex displaying about 60% of the original activity. 3. The subunits were isolated by sucrose-density-gradient centrifugation and studied for the different partial enzyme activities involved in long-chain fatty acid synthesis: malonyl, palmitoyl and acetyl transferase, enoyl reductase and dehydratase were shown to be exclusive functions of the beta chains of the complex, confirming a pentafunctional role of this subunit.     

Developmental specific expression and organelle targeting of the Escherichia coli fabD gene,encoding malonyl coenzyme A-acyl carrier protein transacylase in transgenic rape and tobacco seeds

In both plants and bacteria, de novo fatty acid biosynthesis is catalysed by a type II fatty acid synthetase (FAS) system which consists of a group of eight discrete enzyme components. The introduction of heterologous, i.e. bacterial, FAS genes in plants could provide an alternative way of modifying the plant lipid composition. In this study the Escherichia coli fabD gene, encoding malonyl CoA-ACP transacylase (MCAT), was used as a model gene to investigate the effects of over-producing a bacterial FAS component in the seeds of transgenic plants. Chimeric genes were designed, so as not to interfere with the household activities of fatty acid biosynthesis in the earlier stages of seed development, and introduced into tobacco and rapeseed using the Agrobacterium tumefaciens binary vector system. A napin promoter was used to express the E. coli MCAT in a seed-specific and developmentally specific manner. The rapeseed enoyl-ACP reductase transit peptide was used successfully, as confirmed by immunogold labelling studies, for plastid targeting of the bacterial protein. The activity of the bacterial enzyme reached its maximum (up to 55 times the maximum endogenous MCAT activity) at the end of seed development, and remained stable in mature transgenic seeds. Significant changes in fatty acid profiles of storage lipids and total seed lipid content of the transgenic plants were not found. These results are in support of the notion that MCAT does not catalyse a rate-limiting step in plant fatty acid biosynthesis.     

Co-ordinate regulation of sterol biosynthesis enzyme activity during accumulation of sterols in developing rape and tobacco seed

The activities of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, sterol methyl transferase 1 and sterol acyltransferase, key enzymes involved in phytosterol biosynthesis were shown to be co-ordinately regulated during oilseed rape ( Brassica napus L.) and tobacco ( Nicotiana tabacum L.) seed development. In both plants, enzyme activities were low during the initial stages of seed development, increasing towards mid-maturation where they remained stable for a time, before declining rapidly as the oilseeds reached maturity. During seed development, the level of total sterols increased 12-fold in tobacco and 9-fold in rape, primarily due to an increase in steryl ester production. In both seed tissues, stages of maximum enzyme activity coincided with periods of high rates of sterol production, indicating developmental regulation of the enzymes to be responsible for the increases in the sterol content observed during seed development. Consistent with previous studies the data presented suggest that sterol biosynthesis is regulated by two key steps, although there may be others. The first is the regulation of carbon flux into the isoprenoid pathway to cycloartenol. The second is the flux from cycloartenol to Delta(5)-end-product sterols. The implications of the results in terms of enhancing seed sterol levels by genetic modification are also discussed.     

Expression, purification and characterization of enoyl-ACP reductase II, FabK, from Porphyromonas gingivalis

The rapid rise in bacterial drug resistance coupled with the low number of novel antimicrobial compounds in the discovery pipeline has led to a critical situation requiring the expedient discovery and characterization of new antimicrobial drug targets. Enzymes in the bacterial fatty acid synthesis pathway, FAS-II, are distinct from their mammalian counterparts, FAS-I, in terms of both structure and mechanism. As such, they represent attractive targets for the design of novel antimicrobial compounds. Enoyl-acyl carrier protein reductase II, FabK, is a key, rate-limiting enzyme in the FAS-II pathway for several bacterial pathogens. The organism, Porphyromonas gingivalis, is a causative agent of chronic periodontitis that affects up to 25% of the US population and incurs a high national burden in terms of cost of treatment. P. gingivalis expresses FabK as the sole enoyl reductase enzyme in its FAS-II cycle, which makes this a particularly appealing target with potential for selective antimicrobial therapy. Herein we report the molecular cloning, expression, purification and characterization of the FabK enzyme from P. gingivalis, only the second organism from which this enzyme has been isolated. Characterization studies have shown that the enzyme is a flavoprotein, the reaction dependent upon FMN and NADPH and proceeding via a Ping-Pong Bi-Bi mechanism to reduce the enoyl substrate. A sensitive assay measuring the fluorescence decrease of NADPH as it is converted to NADP(+) during the reaction has been optimized for high-throughput screening. Finally, protein crystallization conditions have been identified which led to protein crystals that diffract x-rays to high resolution.     

A study of the structure-activity relationship for diazaborine inhibition of Escherichia coli enoyl-ACP reductase

Enoyl acyl carrier protein (ACP) reductase catalyses the last reductive step of fatty acid biosynthesis, reducing the enoyl group of a growing fatty acid chain attached to ACP to its acyl product using NAD(P)H as the cofactor. This enzyme is the target for the diazaborine class of antibacterial agents, the biocide triclosan, and one of the targets for the front-line anti-tuberculosis drug isoniazid. The structures of complexes of Escherichia coli enoyl-ACP reductase (ENR) from crystals grown in the presence of NAD+ and a family of diazaborine compounds have been determined. Analysis of the structures has revealed that a mobile loop in the structure of the binary complex with NAD+ becomes ordered on binding diazaborine/NAD+ but displays a different conformation in the two subunits of the asymmetric unit. The work presented here reveals how, for one of the ordered conformations adopted by the mobile loop, the mode of diazaborine binding correlates well with the activity profiles of the diazaborine family. Additionally, diazaborine binding provides insights into the pocket on the enzyme surface occupied by the growing fatty acid chain.     

Interrelationships among human aldo-keto reductases: immunochemical, kinetic and structural properties

We have proposed earlier a three gene loci model to explain the expression of the aldo-keto reductases in human tissues. According to this model, aldose reductase is a monomer of alpha subunits, aldehyde reductase I is a dimer of alpha, beta subunits, and aldehyde reductase II is a monomer of delta subunits. Using immunoaffinity methods, we have isolated the subunits of aldehyde reductase I (alpha and beta) and characterized them by immunocompetition studies. It is observed that the two subunits of aldehyde reductase I are weakly held together in the holoenzyme and can be dissociated under high ionic conditions. Aldose reductase (alpha subunits) was generated from human placenta and liver aldehyde reductase I by ammonium sulfate (80% saturation). The kinetic, structural and immunological properties of the generated aldose reductase are similar to the aldose reductase obtained from the human erythrocytes and bovine lens. The main characteristic of the generated enzyme is the requirement of Li2SO4 (0.4 M) for the expression of maximum enzyme activity, and its Km for glucose is less than 50 mM, whereas the parent enzyme, aldehyde reductase I, is completely inhibited by 0.4 M Li2SO4 and its Km for glucose is more than 200 mM. The beta subunits of aldehyde reductase I did not have enzyme activity but cross-reacted with anti-aldehyde reductase I antiserum. The beta subunits hybridized with the alpha subunits of placenta aldehyde reductase I, and aldose reductase purified from human brain and bovine lens. The hybridized enzyme had the characteristic properties of placenta aldehyde reductase I.     

Vibrio cholerae FabV defines a new class of enoyl-acyl carrier protein reductase

Enoyl-acyl carrier protein (ACP) reductase catalyzes the last step of the fatty acid elongation cycle. The paradigm enoyl-ACP reductase is the FabI protein of Escherichia coli that is the target of the antibacterial compound, triclosan. However, some Gram-positive bacteria are naturally resistant to triclosan due to the presence of the triclosan-resistant enoyl-ACP reductase isoforms, FabK and FabL. The genome of the Gram-negative bacterium, Vibrio cholerae lacks a gene encoding a homologue of any of the three known enoyl-ACP reductase isozymes suggesting that this organism encodes a novel fourth enoyl-ACP reductase isoform. We report that this is the case. The gene encoding the new isoform, called FabV, was isolated by complementation of a conditionally lethal E. coli fabI mutant strain and was shown to restore fatty acid synthesis to the mutant strain both in vivo and in vitro. Like FabI and FabL, FabV is a member of the short chain dehydrogenase reductase superfamily, although it is considerably larger (402 residues) than either FabI (262 residues) or FabL (250 residues). The FabV, FabI and FabL sequences can be aligned, but only poorly. Alignment requires many gaps and yields only 15% identical residues. Thus, FabV defines a new class of enoyl-ACP reductase. The native FabV protein has been purified to homogeneity and is active with both crotonyl-ACP and the model substrate, crotonyl-CoA. In contrast to FabI and FabL, FabV shows a very strong preference for NADH over NADPH. Expression of FabV in E. coli results in markedly increased resistance to triclosan and the purified enzyme is much more resistant to triclosan than is E. coli FabI.     

Resolution of distinct membrane-bound enzymes from Enterobacter cloacae SLD1a-1 that are responsible for selective reduction of nitrate and selenate oxyanions

Enterobacter cloacae SLD1a-1 is capable of reductive detoxification of selenate to elemental selenium under aerobic growth conditions. The initial reductive step is the two-electron reduction of selenate to selenite and is catalyzed by a molybdenum-dependent enzyme demonstrated previously to be located in the cytoplasmic membrane, with its active site facing the periplasmic compartment (C. A. Watts, H. Ridley, K. L. Condie, J. T. Leaver, D. J. Richardson, and C. S. Butler, FEMS Microbiol. Lett. 228:273-279, 2003). This study describes the purification of two distinct membrane-bound enzymes that reduce either nitrate or selenate oxyanions. The nitrate reductase is typical of the NAR-type family, with alpha and beta subunits of 140 kDa and 58 kDa, respectively. It is expressed predominantly under anaerobic conditions in the presence of nitrate, and while it readily reduces chlorate, it displays no selenate reductase activity in vitro. The selenate reductase is expressed under aerobic conditions and expressed poorly during anaerobic growth on nitrate. The enzyme is a heterotrimeric (alphabetagamma) complex with an apparent molecular mass of approximately 600 kDa. The individual subunit sizes are approximately 100 kDa (alpha), approximately 55 kDa (beta), and approximately 36 kDa (gamma), with a predicted overall subunit composition of alpha3beta3gamma3. The selenate reductase contains molybdenum, heme, and nonheme iron as prosthetic constituents. Electronic absorption spectroscopy reveals the presence of a b-type cytochrome in the active complex. The apparent Km for selenate was determined to be approximately 2 mM, with an observed Vmax of 500 nmol SeO4(2-) min(-1) mg(-1) (kcat, approximately 5.0 s(-1)). The enzyme also displays activity towards chlorate and bromate but has no nitrate reductase activity. These studies report the first purification and characterization of a membrane-bound selenate reductase.     

Kinetic and nuclear magnetic resonance study of the interaction of NADP+ and NADPH with chicken liver fatty acid synthase

The ionic strength dependence of the second-order rate constant for the association of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and chicken liver fatty acid synthase was determined. This rate constant is 7.2 X 10(7) M-1 s-1 at zero ionic strength and 25 degrees C; the effective charge at the cofactor binding sites is +0.8. The conformations of nicotinamide adenine dinucleotide phosphate (NADP+) and NADPH bound to the beta-ketoacyl and enoyl reductase sites were determined from transferred nuclear Overhauser effect measurements. Covalent modification of the enzyme with pyridoxal 5'-phosphate abolished cofactor binding at the enoyl reductase site; this permitted the cofactor conformations at the beta-ketoacyl and enoyl reductase sites to be distinguished. For NADP+ bound to the enzyme, the conformation of the nicotinamide-ribose bond is anti at the enoyl reductase site and syn at the beta-ketoacyl reductase site; the adenine-ribose bond is anti, and the sugar puckers are C3'-endo. Nicotinamide-adenine base stacking was not detected. Structural models of NADP+ at the beta-ketoacyl and enoyl reductase sites were constructed by using the distances calculated from the observed nuclear Overhauser effects. Because of the overlap of the resonances of several nonaromatic NADPH protons with the resonances of HDO and ribose protons, less extensive structural information was obtained for NADPH bound to the enzyme. However, the conformations of NADPH bound to the two reductases are qualitatively the same as those of NADP+, except that the nicotinamide moiety of NADPH is closer to being fully anti at the enoyl reductase site.     

Correlation of enzymatic activities and aggregation state in chicken liver fatty acid synthase

The relationships between the aggregation state and the enzymatic activities of chicken liver fatty acid synthase have been explored by monitoring the changes in light scattering, fluorescence, and the overall, beta-ketoacyl synthase, beta-ketoacyl reductase and enoyl reductase activities during dissociation and reassociation of the enzyme. The data obtained indicate that the enzyme dissociates at low temperature in both 0.1 M potassium phosphate (pH 7.0), 1 mM EDTA, and 5 mM Tris(hydroxymethyl)aminomethane, 35 mM glycine (pH 8.3) and 1 mM EDTA, but the extent of dissociation is less in the phosphate buffer. The assay conditions influence the assessment of the degree of dissociation and association: high temperatures, phosphate (high salt), NADPH and acetoacetyl-coenzyme A promote association of the monomeric enzyme, whereas dilution in the Tris-glycine buffer (low salt) and low temperature promote dissociation. Both the rate and extent of association and dissociation are altered by substrates. The monomeric enzyme does not possess beta-ketoacyl synthase and beta-ketoacyl reductase activities. Results obtained with the 1,3-dibromo-2-propanone cross-linked enzyme, which lacks beta-ketoacyl synthase activity, indicate that the NADPH-binding site of beta-ketoacyl reductase is disrupted at low ionic strength. In contrast, changes in ionic strength have little effect on the enoyl reductase activity. The dimer is stabilized by both electrostatic and hydrophobic interactions, with the former being of special importance for maintenance of the beta-ketoacyl reductase active site. site.     

Biochemical properties of short- and long-chain rat liver microsomal trans-2-enoyl coenzyme A reductase

This study describes the biochemical properties of the rat hepatic microsomal NADPH-specific short-chain enoyl CoA reductase and NAD(P)H-dependent long-chain enoyl CoA reductase. Of the substrates tested, crotonyl CoA and trans-2-hexenoyl CoA are reduced by the short-chain reductase only in the presence of NADPH. The trans-2-octenoyl CoA and trans-2-decenoyl CoA appear to undergo reduction to octanoate and decanoate, respectively, catalyzed by both enzymes; 64% conversion of the C8:1 is catalyzed by the short-chain reductase, while 36% conversion is catalyzed by the long-chain enzyme. For the C10:1 substrate, 45% is converted by the short-chain reductase, while 55% is reduced by the long-chain reductase. trans-2-Hexadecenoyl CoA is a substrate for the long-chain enoyl CoA reductase only. Reduction of C4 and C6 enoyl CoA's was unaffected by bovine serum albumin (BSA), whereas BSA markedly stimulated the conversion of C10 and C16 enoyl CoA's to their respective saturated product. Reduction rates as a function of microsomal protein concentration, incubation time, pH, and cofactors are reported including the apparent Km and Vmax for substrates and cofactors. In general, the apparent Km's for the substrates ranged from 19 to 125 microM. The apparent Vmax for the short-chain enoyl CoA reductase was greatest with trans-2-hexenoyl CoA, having a turnover of 65 nmol/min/mg microsomal protein, while the apparent Vmax for the long-chain enzyme was greatest with trans-2-hexadecenoyl CoA, having a turnover of 55 nmol/min/mg microsomal protein. With respect to electron input, NADPH-cytochrome P-450 reductase, either alone, mixed with phospholipid, or incorporated into phospholipid vesicles, possessed no enoyl CoA reductase activity. Cytochrome c did not affect the NADPH-dependent conversion of the trans-2-enoyl CoA. In addition, anti-NADPH-cytochrome P-450 reductase IgG did not inhibit the reduction of trans-2-hexadecenoyl CoA in hepatic microsomes. Finally, the NADPH-specific short-chain and NAD(P)H-dependent long-chain enoyl CoA reductases were solubilized and completely separated from NADPH-cytochrome P-450 reductase by employing DE-52 column chromatography. These studies demonstrate the noninvolvement of NADPH-cytochrome P-450 reductase in either the short-chain (13) or long-chain enoyl CoA reductase system. Thus, the role of NADPH-cytochrome P-450 reductase in the microsomal elongation of fatty acids appears to be at the level of the first reduction step.     

Crystallographic analysis of triclosan bound to enoyl reductase

Molecular genetic studies with strains of Escherichia coli resistant to triclosan, an ingredient of many anti-bacterial household goods, have suggested that this compound works by acting as an inhibitor of enoyl reductase (ENR) and thereby blocking lipid biosynthesis. We present structural analyses correlated with inhibition data, on the complexes of E. coli and Brassica napus ENR with triclosan and NAD(+) which reveal how triclosan acts as a site-directed, picomolar inhibitor of the enzyme by mimicking its natural substrate. Elements of both the protein and the nucleotide cofactor play important roles in triclosan recognition, providing an explanation for the factors controlling its tight binding to the enzyme and for the emergence of triclosan resistance.     

Induction,purification and characterisation of acyl-ACP thioesterase from developing seeds of oil seed rape (Brassica napus)

The level of two thioesterases, acyl-CoA thioesterase and acyl-ACP thioesterase was determined during seed maturation in oil seed rape. Both thioesterase activities rose markedly prior to the onset of lipid accumulation, but the induction kinetics suggest that the activities reside on distinct polypeptides. Acyl-ACP thioesterase (EC 3.1.2.14) was purified 2000-fold using a combination of ion exchange, ACP-affinity chromatogr aphy, chromatofocusing and gel filtration. Using native gel electrophoresis, and assays for enzymic activity, two polypeptides were identified on SDS-PAGE as associated with the activity. Cleveland mapping of these polypeptides, of 38 kDa component and 33 kDa respectively, demonstrated that they are related. An antibody was prepared against the 38 kDa component, and this also recognises the 33 kDa polypeptide in highly purified preparations. Western blotting of a crude extract identifies one band at 38 kDa consistent with the 33 kDa component being a degradation product generated during purification. The native molecule has a Mr of 70 kDa indicating a dimeric structure. The enzyme has a pH optimum of 9.5 and shows strong preference for oleoyl-ACP as substrate. The intact enzyme has an N-terminus blocked to protein sequencing. We also found that two other polypeptides co-purify with acyl-ACP thioesterase under native conditions. The N-terminal amino-acid sequence of these polypeptides is shown and their possible identity is discussed.     

Exploring the interaction energies for the binding of hydroxydiphenyl ethers to enoyl-acyl carrier protein reductases

It is now well established that the potent anti-microbial compound, triclosan, interrupts the type II fatty acid synthesis by inhibiting the enzyme enoyl-ACP reductase in a number of organisms. Existence of a high degree of similarity between the recently discovered enoyl-ACP reductase from P. falciparum and B. napus enzyme permitted building of a satisfactory model for the former enzyme that explained some of the key aspects of the enzyme such as its specificity for binding to the cofactor and the inhibitor. We now report the interaction energies between triclosan and other hydroxydiphenyl ethers with the enzymes from B. napus, E. coli and P. falciparum. Examination of the triclosan-enzyme interactions revealed that subtle differences exist in the ligand binding sites of the enzymes from different sources i.e., B. napus, E. coli and P. falciparum. A comparison of their binding propensities thus determined should aid in the design of effective inhibitors for the respective enzymes.     

Purification and characterizations of beta-Ketoacyl-[acyl-carrier-protein] reductase, beta-hydroxyacyl-[acyl-carrier-protein] dehydrase, and enoyl-[acyl-carrier-protein] reductase from Spinacia oleracea leaves

β-Ketoacyl-[acyl-carrier-protein] (ACP) reductase, β-hydroxyacyl-ACP dehydrase, and enoyl-ACP reductase have been purified to homogeneity from extracts of spinach leaves. Based on sodium dodecyl sulfate-polyacrylamide gel eletrophoresis studies, the monomeric molecular weights of the β-ketoacyl-ACP reductase, β-hydroxyacyl-ACP dehydrase, and enoyl-ACP reductase were 24,200, 19,000, and 32,500, respectively, and by gel filtration, their molecular weights were 97,000, 85,000, and 115,000, respectively, suggesting that these three enzymes exist as tetramers. The β-ketoacyl-ACP reductase, the β-hydroxyacyl-ACP dehydrase, and the enoyl-ACP reductase contained two, one, and two cystein residues per monomer. β-Ketoacyl-ACP reductase preferably utilized NADPH as the reductant, whereas enoyl-ACP reductase was absolutely specific to NADH. β-Ketoacyl-ACP reductase reversibly catalyzed the reduction of acetoacetyl-ACP to d-β-hydroxybutyryl-ACP and β-hydroxyacyl-ACP dehydrase catalyzed the dehydration of d-β-hydroxyacyl-ACP to 2-enoyl-ACP. Both β-hydroxyacyl-ACP dehydrase and enoyl-ACP reductase were active with 2-enoyl-ACPs having chain lengths from C₄ to C₁₆, with 2-hexenoyl-ACP and 2-octenoyl-ACP being the most effective substrate. CoA esters served as substrates with the β-ketoacyl-ACP reductase and the enoyl-ACP reductase but were inert with β-hydroxyacyl-ACP dehydrase. These enzymes were inhibited by p-chloromercuribenzoate but not by N-ethylmaleimide.     

Inhibition of InhA, the enoyl reductase from Mycobacterium tuberculosis, by triclosan and isoniazid

Structural and genetic studies indicate that the antibacterial compound triclosan, an additive in many personal care products, is an inhibitor of EnvM, the enoyl reductase from Escherichia coli. Here we show that triclosan specifically inhibits InhA, the enoyl reductase from Mycobacterium tuberculosis and a target for the antitubercular drug isoniazid. Binding of triclosan to wild-type InhA is uncompetitive with respect to both NADH and trans-2-dodecenoyl-CoA, with K(i)' values of 0.22+/-0.02 and 0.21+/-0.01 microM, respectively. Replacement of Y158, the catalytic tyrosine residue, with Phe, reduces the affinity of triclosan for the enzyme and results in noncompetitive inhibition, with K(i) and K(i)' values of 36+/-5 and 47+/-5 microM, respectively. Consequently, the Y158 hydroxyl group is important for triclosan binding, suggesting that triclosan binds in similar ways to both InhA and EnvM. In addition, the M161V and A124V InhA mutants, which result in resistance of Mycobacterium smegmatis to triclosan, show significantly reduced affinity for triclosan. Inhibition of M161V is noncompetitive with K(i)' = 4.3+/-0.5 microM and K(i) = 4.4+/-0.9 microM, while inhibition of A124V is uncompetitive with K(i)' = 0. 81 +/- 0.11 microM. These data support the hypothesis that the mycobacterial enoyl reductases are targets for triclosan. The M161V and A124V enzymes are also much less sensitive to isoniazid compared to the wild-type enzyme, indicating that triclosan can stimulate the emergence of isoniazid-resistant enoyl reductases. In contrast, I47T and I21V, two InhA mutations that occur in isoniazid-resistant clinical isolates of M. tuberculosis, show unimpaired inhibition by triclosan, with uncompetitive inhibition constants (K(i)') of 0.18+/-0.01 and 0.12+/- 0.01 microM, respectively. The latter result indicates that InhA inhibitors targeted at the enoyl substrate binding site may be effective against existing isoniazid-resistant strains of M. tuberculosis.