Triclosan offers protection against blood stages of. Crystal structures and kinetic properties of enoyl acyl carrier protein reductase i from candidatus liberibacter asiaticus ling jiang,1 zengqiang gao,2 yanhua li,2 shennan wang,1 and yuhui dong2 1ministry of education key laboratory of plant biology, department of horticulture and forestry, huazhong agricultural univer sity, wuhan 430070, china. Biosynthesis of lovastatin uses an iterative type i polyketide synthase pks. Here we investigated the factors that direct catalysis in the enoyl thioester reductase etr1p.
Conformational changes caused by inhibition of inha, the enoyl reductase from mycobacterium tuberculosis. Enoylacylcarrierprotein reductase nadph, aspecific. The unique enzyme enoyl acp reductase fabi of plasmodium commits the final reduction step during the fatty acid elongation process, therefore representing an excellent target for the development of novel antiplasmodial agents surolia and surolia, 2001. Steadystate kinetic parameters for trans2,trans4hexadienoylcoa and 5phenyltrans2,trans4pentadienoylcoa were determined. Biochemical and structural basis of triclosan resistance. Pdf files are great for protecting the integrity of a document, but they can be a hassle when it comes time to print antimicobcterianos. A cis acting enoyl reductase er catalytic domain was isolated from a. Mechanisms of isoniazid resistance in mycobacterium.
In fabi the triad is comprised of tyr 146, tyr 156, and lys 163, whereas in inha, the m. Smith1,5 1life sciences institute, university of michigan, 210 washtenaw avenue, ann arbor, mi 48109, usa. Genes of the facultative intracellular pathogen listeria monocytogenes encode two functional enoyl acyl carrier protein isoforms based on their ability to complement the temperaturesensitive growth. The second round of extension and modi cationisthesame. Inha has been shown to be the primary target of isoniazid inh, one of the oldest synthetic antitubercular drugs. Oct 28, 2019 enoyl acyl carrier protein reductase enr catalyzes the last reduction step in the bacterial type ii fatty acid biosynthesis cycle. Stereochemical considerations are important in both isomerism and studies of the mechanisms of chemical reactions. Recent studies suggest that enr is also the target for the broadspectrum biocide triclosan. It is not only a key enzyme in the pathway, but also the most diverse. Inactivation of the inhaencoded fatty acid synthase ii.
Peter r donald, helen mcilleron, in tuberculosis, 2009. Fabi is a member of the short chain alcohol dehydrogenase reductase family. Stereochemistry subtle differences in spatial arrangements. A conserved threonine prevents selfintoxication of enoyl.
When an enoylreductase enzyme of a modular polyketide synthase reduces a. Nicotinamide adenine dinucleotide nad is a cofactor that is central to metabolism. Introduction to stereochemistry compounds that are not superimposable with their mirror image are called chiral in greek, chiral means handed 3methylhexane is a chiral molecule. Characterization of pseudomonas aeruginosa enoylacyl. Further considerations on the reaction mechanism indicated that crotonylcoa carboxylase reductase may have evolved from enoyl coa reductases. Structural basis for cyclopropanation by a unique enoyl.
This enzyme is the target of the antibacterial drug triclosan and has been the subject of extensive studies for the past 20 years. Cyperin is a natural diphenyl ether phytotoxin produced by several fungal plant pathogens. Using high throughput inhibitor screens with a combinatorial library, we have identified two novel classes of compounds with activity against the m. The target of activated isoniazid is the nadhdependent enoyl acyl carrier protein reductase, coded for by the inha gene, which promotes formation of long fatty acyl chains of mycolic acids and, consequently, bacilli on which isoniazid. Mycobacterium tuberculosis enoylacyl carrier protein. The enoyl acyl carrier protein acp reductase catalyses the last step in the elongation cycle of fatty acids. Spectrum antibacterial drug seol 2019 bulletin of the korean chemical society wiley online library. This enzyme catalyzes the nadphdependent reduction of 2,4dienoylcoenzyme a coa thiolesters to the resulting trans3 enoyl coa. Enoyl acyl carrier protein reductase catalyzes the last step in each elongation cycle of type ii bacterial fatty acid synthesis and is a key regulatory protein in bacterial fatty acid synthesis. Crystal structures and kinetic properties of enoylacyl. Recombinant inha proteins with defined single amino acid replacements were expressed in escherichia coli and purified to homogeneity. At high concentrations, this metabolite inhibits protoporphyrinogen oxidase, a key enzyme in porphyrin synthesis.
Stereochemistry an introduction pdf 40p this note covers the following topics. Stereochemistry of tetrahedral carbons, stereoisomers stereoisomers, stereocenter, chiral, enantiomers, racemic mixture, configuration of stereocenters, molecules with multiple stereocenters, tartaric acid and enantiomers. Yet, this is the most common convention used, and it is the convention we adopt in this book. The enoyl acyl carrier protein reductase enr is the last enzyme in the fatty acid elongation cycle. This superfamily is characterized by a conserved triad of active site residues. Prediction and manipulation of the stereochemistry of. The enoyl acyl carrier protein reductase enr is involved in bacterial fatty acid biosynthesis and is the target of the antibacterial diazaborine compounds and the frontline antituberculosis drug isoniazid. The enzyme is a component of the type ii dissociated fatty acid synthase in bacteria and catalyzes the terminal reaction in the fatty acid elongation cycle.
Structural basis for cyclopropanation by a unique enoylacyl carrier protein reductase dheeraj khare,1 wendi a. Further considerations on the reaction mechanism indicated that crotonylcoa carboxylasereductase may have evolved from enoylcoa reductases. Stereochemistry projecting away from the viewer projecting toward the viewer hydrogens projecting toward the viewer the convention used in this book it moves further away. Enoylacyl carrier protein reductase i fabi is essential. Pdf carboxylation mechanism and stereochemistry of crotonyl.
Inha, the nadhdependent 2trans enoyl acp reductase enzyme from mycobacterium tuberculosis mtb, is involved in the biosynthesis of mycolic acids, the hallmark of mycobacterial cell wall. Jun 02, 2009 our findings led to a reassessment of the stereospecificity of enoyl thioester reductases and related enzymes with respect to their amino acid sequence, revealing a general pattern of stereospecificity that allows the prediction of the stereochemistry of the hydride transfer for enoyl reductases of unknown specificity. Surprisingly the results of two different assays showed that the isolated er. A cisacting enoyl reductase er catalytic domain was isolated from a fungal highly reducing iterative polyketide synthase hripks for the first time and studied in vitro. In intact sqtks the er domain sets the stereochemistry of the two methylated positions, presumably via a stereoselective reprotonation at the 2position of an enolate intermediate 35, itself created by transfer of hydride to the 3carbon of the enoyl substrate. The assay was initiated by the addition of crotonylcoa. Substrate selectivity of an isolated enoyl reductase. Structure of acyl carrier protein bound to fabi, the fasii. Structural basis and mechanism of enoyl reductase inhibition. When extra copies of love are added, lovastatin production increases, and when either of these genes are rendered inactive via mutations, lovastatin and its inter.
Compounds that are superimposable with their mirror image are called achiral. A cisacting enoyl reductase er catalytic domain was isolated from a fungal highly reducing iterative polyketide synthase hripks for the. The stereochemical course of reduction of crotonyl coa by the novel crotonyl coa reductase ccr of streptomyces collinus was determined using a radiochemical assay. However, unlike its herbicide structural analogs, the mode of action of cyperin is not light dependent, causing loss of membrane integrity in the dark. Lovc is a transacting enoyl reductase er that specifically reduces three out of eight possible polyketide intermediates during lovastatin biosynthesis. The two functional enoylacyl carrier protein reductases. Reduction of the growing polyketide chain, before the dielsalder reaction, can be mediated by the pkss using the ketoreductase kr, dehydratase dh, and enoyl reductase er 4 as observed in solanapyrones biosynthesis.
A majority of the enzymes involved in the fasii cycle are relatively conserved. Substrate selectivity of an isolated enoyl reductase catalytic domain. The stereochemical course of the enoyl reduction catalyzed by fatty acid synthetase was investigated using the enzymes from rat liver and brevibacterium ammoniagenes. Lovastatin is an important statin prescribed for the treatment and prevention of cardiovascular diseases. Two genes, love and lovh, have homology with transcription factors. Carboxylation mechanism and stereochemistry of crotonylcoa carboxylase reductase article pdf available in proceedings of the national academy of sciences 10622. The tubes containing high enzymatic activities were combined, the concentration of phosphate was adjusted to 0. Enzymes are highly specific biocatalysts, yet they can promote unwanted side reactions.
Sch 511 biosynthesis of fatty acids university of nairobi. Inh is a prodrug which is biologically activated by the mtb catalaseperoxidase. The synthesis of odd numbered fatty acids involve the use of the same extender unit, malonyl sacp, with an odd numbered starter unit. Acyl carrier protein reductase fabi proposes a putative inhibitor binding pocket. Biosyntheses of antibiotic a26771b by penicillium turbatum. Hale,2,7 ashootosh tripathi,1 liangcai gu,1,3,8 david h. Functional analysis of the fusarielin biosynthetic gene cluster. Partially hydrogenated cooking oils are the primary source of transfats. Enoylacyl carrier protein reductase enr catalyzes the nadhdependent stereospecific reduction of. Despite the large number of reports describing the biochemistry of this enzyme, there have been no studies that. Pkss results in the inversion of alcohol stereochemistry. Such transacting ers have been reported across a variety of other fungal pks enzymes as a strategy in nature to diversify polyketides.
Unlike fatty acid biosynthesis, however, in which each successive chain elongation step is followed by a fixed sequence of keto reduction, dehydration and enoyl. Despite this complexity, a preponderance of evidence implicates inha, which codes for an enoyl acyl carrier protein reductase of the fatty acid synthase ii fasii, as the primary target of inh. This may be useful for protein engineering of enoyl. Nadh, nadph, and reduced flavin mononucleotide fmnh 2 function as coenzymes in this reduction reaction. In this work, multicopy plasmids expressing either inha or kasa genes were transformed into m. The biosynthesis of fatty acids that possesses an odd number of carbon atoms is essentially similar to the biosynthesis of their even numbered counter parts.
Enoyl acyl carrier protein enoyl acp reductases enrs reduce trans2 enoyl acps to the fully saturated acp species in the last step of the elongation cycle in the synthesis of bacterial and plant fatty acids 7, 1114 fig. Transfats are associated with increased risk of cardiovascular disease and are targeted for removal from the food supply. The deuteriumlabeled fatty acids thus produced were subjected to. The mechanism of dienoylcoa reduction by 2,4dienoylcoa. Inha, the enoylthioester reductase from mycobacterium tuberculosis. The stereochemistry of complex polyketide biosynthesis citeseerx. Enr is an attractive target for narrowspectrum antibacterial drug discovery because of its essential role in metabolism and its sequence conservation across many bacterial species. Structural basis for cyclopropanation by a unique enoylacyl. Stereochemistry is defined as the study of the threedimensional structure of molecules. Steadystate kinetic parameters for trans2,trans4hexadienoylcoa and 5phenyltrans2,trans4pentadienoylcoa were determined and. The enoyl thioester reductase inha catalyzes an essential step in fatty acid. Bacterial enoyl acyl carrier protein acp reductase has been con.
Enoyl coa hydratase ech or crotonase is an enzyme that hydrates the double bond between the second and third carbons on 2transcis enoyl coa ech is essential to metabolizing fatty acids in beta oxidation to produce both acetyl coa and energy in the form of atp ech of rats is a hexameric protein this trait is not universal, but human enzyme is also hexameric, which leads to the. The reaction was shown to proceed with transfer of the hydrogen from the pro4s position of nadph to the re face of the. It disrupts the integrity of mycobacterial cell wall via inhibiting the biosynthesis of mycolic acids, a long chain fatty acids containing component of the cell envelope. The enzyme enoyl acp reductase also called fabi in bacteria is an essential member of the fatty acid synthase ii pathway in plants and bacteria. Introduction to stereochemistry structural constitutional isomers compounds of the same molecular formula with different connectivity structure, constitution conformational isomers compounds of the same structure that differ in rotation around one or more single bonds configurational isomers or stereoisomers compounds of the same structure that differ in one or more. Screening and sequence analyses of the triclosan resistome from the soil metagenome identified a variety of triclosan. This may be useful for protein engineering of enoyl reductases and their application in biocatalysis. However, inh treatment of mycobacterium tuberculosis causes the accumulation of hexacosanoic acid c 26. Our findings led to a reassessment of the stereospecificity of enoyl thioester reductases and related enzymes with respect to their amino acid sequence, revealing a general pattern of stereospecificity that allows the prediction of the stereochemistry of the hydride transfer for enoyl reductases of unknown specificity. Pdf conformational changes in 2 trans enoylacp coa. In the screening of inhibitors of staphylococcus aureus enoyl acp reductase fabi, we found that a methanol extract of leaves of morus alba l. Enoyl reductase enr, an enzyme involved in fatty acid biosynthesis, is the target for antibacterial diazaborines and the frontline antituberculosis drug isoniazid. A mechanism of drug action revealed by structural studies.
One nucleotide contains an adenine nucleobase and the other nicotinamide. A pathogenic fungi diphenyl ether phytotoxin targets plant. Common themes in redox chemistry emerge from the xray. Starch and cellulose are two more examples of molecules that have the same connectivity, but different 3d arrangement of atoms. Stereochemistry some definitions with examples presenting stereo structures goes back in the plane of the paper comes forward chiral center representaiton comes forward goes back goes back dotted line wedge fischer projection stereoisomersisomers that are different because of their orientation in space. Enoyl acyl carrier protein enoyl acp reductase catalyzes the last step of the elongation cycle in the synthesis of bacterial fatty acids. Stereochemical course of enoyl reduction catalyzed by. Determination and interpretation of the novel stereochemical course of reaction catalyzed by crotonyl coa reductase from streptomyces collinus. The extender condenses with an acyl chain that is thioesterlinked to the active site cysteine of a ketosynthase ks. Journal of the american chemical society 1997, 119 , 29732979. Linking diversity in evolutionary origin and stereospecificity for enoyl thioester reductases. Erb a,1, volker brecht b, georg fuchs a, michael mu. Other names in common use include acylacp dehydrogenase, enoyl acyl carrier protein reduced nicotinamide adenine, dinucleotide phosphate reductase, nadph 2 enoyl co a reductase, enoyl acp reductase, and enoyl acylcarrierprotein reductase nadph2, aspecific. Carboxylation mechanism and stereochemistry of crotonyl.
Enoyl acyl carrier protein reductase enr, a key type ii enzyme, has been repeatedly validated as an effective antimicrobial target. Chapterii towards the synthesis of the macrocyclic core of pladienolides a and b 41 understood way. The enoylacylcarrierprotein reductases fabi and fabl. One of these, the acyclic 4s isomer of isoniazidnad, targets the inhaencoded enoyl acp reductase, an enzyme essential for mycolic acid biosynthesis in mycobacterium tuberculosis.
Physiologically, fabi is an important enzyme because i reduction of enoyl acp derivatives is thought to regulate the ratio of saturated to unsaturated fatty acids and to coor. Transfer of acetylcoa from mitochondria to cytosol. Inha has been shown to be the primary target of isoniazid. Furthermore, the normal reduc tive activities of a fatty acid synthase ketoreductase kr, dehy dratase dh, and enoylreductase er activities. An entire pks consists of a series of modules that are housed within large multienzyme polypeptides. Escherichia colihas a single enr encoded by the essential fabigene 7, 12. The er shows broad substrate selectivity, reducing enoyl species including both. Found in all living cells, nad is called a dinucleotide because it consists of two nucleotides joined through their phosphate groups. Stereochemistry an introduction pdf 40p download book. Pdf carboxylation mechanism and stereochemistry of. Deuteriumlabeled fatty acids were synthesized by incubating the synthetases with either 4r42 h 2 or 4s42 h 1nadph. Analysis of the structures of complexes of escherichia coli enr with nicotinamide adenine dinucleotide and either thienodiazaborine or benzodiazaborine revealed the formation of a covalent bond between the 2.
Inhibitor binding studies on enoyl reductase reveal conformational changes related to substrate recognition. This may be useful for protein engineering of enoyl reductases and their. The er from the squalestatin tetraketide synthase forms a discrete dimeric protein in solution. The chemical mechanism of the 2,4dienoylcoa reductase ec 1. This transfer represents the first exception to the observation that enoyl. Mutants in the structural gene of the inhaencoded nadhdependent 2trans enoyl acyl carrier protein reductase were identified from isoniazidresistant clinical isolates of mycobacterium tuberculosis. Pdf inhibitor binding studies on enoyl reductase reveal. Directed mutagenesis alters the stereochemistry of. Structure article structural basis for cyclopropanation by a unique enoyl acyl carrier protein reductase dheeraj khare,1 wendi a.
Biochemical and structural insights concerning triclosan. Acyl carrier protein reductase fabi from acinetobacter baumannii as a target for broad. King chapter 5 stereochemistry stereoisomers are compounds that have the same atom connectivity but a different arrangement of atoms in space. The stereochemistry of double bonds in dietary fats fatty acid esters of glycerol is important. Pdf chemo and stereoselective reductions are important reactions in chemistry. A frequently occurring module catalyzes the incorporation of propionate, and reduction of the bketone group to an alcohol. Enoylacp reductase definition of enoylacp reductase by. The enterococcus faecalis genome contains two genes annotated as enoyl acp reductases, a fabitype enoyl acp reductase and a fabktype enoyl acp reductase. The growing interest in enrs is mainly due to the fact that a variety of both synthetic and natural antibacterial compounds are shown to. We report that expression of either of the two proteins restores growth of an escherichia. Characterization of proteinligand binding interactions of.
Carboxylation mechanism and stereochemistry of crotonylcoa carboxylasereductase article pdf available in proceedings of the national academy of sciences 10622. The stereochemistry of ketoreduction sciencedirect. A smaller hydrophobicpacking interface is observed between helices ca51 and or53 with additional hydrogenbonding contacts. Many workers have turned to a simple dashed line instead. Unlike most enzymes in this essential pathway, enr displays an unusual diversity among organisms. Carboxylation mechanism and stereochemistry of crotonylcoa. Enoyl acyl carrier protein acp reductase enr catalyzes the last step of the bacterial type ii fatty acid synthesis fasii cycle to reduce the enoyl acp to fully saturated acylacp fig.
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