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. One nucleotide contains an adenine nucleobase and the other nicotinamide. Structure of acyl carrier protein bound to fabi, the fasii. 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. 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. Triclosan offers protection against blood stages of.
The extender condenses with an acyl chain that is thioesterlinked to the active site cysteine of a ketosynthase ks. Biochemical and structural insights concerning triclosan. Enzymes are highly specific biocatalysts, yet they can promote unwanted side reactions. Screening and sequence analyses of the triclosan resistome from the soil metagenome identified a variety of triclosan. Unlike fatty acid biosynthesis, however, in which each successive chain elongation step is followed by a fixed sequence of keto reduction, dehydration and enoyl. When an enoylreductase enzyme of a modular polyketide synthase reduces a. 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. A smaller hydrophobicpacking interface is observed between helices ca51 and or53 with additional hydrogenbonding contacts. Further considerations on the reaction mechanism indicated that crotonylcoa carboxylase reductase may have evolved from enoyl coa reductases. Fabi is a member of the short chain alcohol dehydrogenase reductase family. Stereochemistry of tetrahedral carbons, stereoisomers stereoisomers, stereocenter, chiral, enantiomers, racemic mixture, configuration of stereocenters, molecules with multiple stereocenters, tartaric acid and enantiomers.
Deuteriumlabeled fatty acids were synthesized by incubating the synthetases with either 4r42 h 2 or 4s42 h 1nadph. The stereochemistry of double bonds in dietary fats fatty acid esters of glycerol is important. Determination and interpretation of the novel stereochemical course of reaction catalyzed by crotonyl coa reductase from streptomyces collinus. Surprisingly the results of two different assays showed that the isolated er. Sch 511 biosynthesis of fatty acids university of nairobi. This enzyme is the target of the antibacterial drug triclosan and has been the subject of extensive studies for the past 20 years. Furthermore, the normal reduc tive activities of a fatty acid synthase ketoreductase kr, dehy dratase dh, and enoylreductase er activities. Unlike most enzymes in this essential pathway, enr displays an unusual diversity among organisms. This superfamily is characterized by a conserved triad of active site residues. 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. A cisacting enoyl reductase er catalytic domain was isolated from a fungal highly reducing iterative polyketide synthase hripks for the. 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.
Stereochemistry an introduction pdf 40p download book. Starch and cellulose are two more examples of molecules that have the same connectivity, but different 3d arrangement of atoms. The growing interest in enrs is mainly due to the fact that a variety of both synthetic and natural antibacterial compounds are shown to. Yet, this is the most common convention used, and it is the convention we adopt in this book. Enoyl acyl carrier protein reductase enr, a key type ii enzyme, has been repeatedly validated as an effective antimicrobial target. Compounds that are superimposable with their mirror image are called achiral.
Structural basis for cyclopropanation by a unique enoylacyl. Nadh, nadph, and reduced flavin mononucleotide fmnh 2 function as coenzymes in this reduction reaction. Found in all living cells, nad is called a dinucleotide because it consists of two nucleotides joined through their phosphate groups. The stereochemical course of reduction of crotonyl coa by the novel crotonyl coa reductase ccr of streptomyces collinus was determined using a radiochemical assay. 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. A majority of the enzymes involved in the fasii cycle are relatively conserved. Here we investigated the factors that direct catalysis in the enoyl thioester reductase etr1p. King chapter 5 stereochemistry stereoisomers are compounds that have the same atom connectivity but a different arrangement of atoms in space. Inha has been shown to be the primary target of isoniazid inh, one of the oldest synthetic antitubercular drugs. Nicotinamide adenine dinucleotide nad is a cofactor that is central to metabolism. 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. 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.
Partially hydrogenated cooking oils are the primary source of transfats. The enoyl acyl carrier protein reductase enr is the last enzyme in the fatty acid elongation cycle. The enterococcus faecalis genome contains two genes annotated as enoyl acp reductases, a fabitype enoyl acp reductase and a fabktype enoyl acp reductase. 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. Stereochemistry an introduction pdf 40p this note covers the following topics. 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. Enoylacyl carrier protein reductase i fabi is essential. The er from the squalestatin tetraketide synthase forms a discrete dimeric protein in solution. Carboxylation mechanism and stereochemistry of crotonylcoa carboxylase reductase article pdf available in proceedings of the national academy of sciences 10622.
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. The second round of extension and modi cationisthesame. Bacterial enoyl acyl carrier protein acp reductase has been con. Using high throughput inhibitor screens with a combinatorial library, we have identified two novel classes of compounds with activity against the m.
Stereochemistry is defined as the study of the threedimensional structure of molecules. Steadystate kinetic parameters for trans2,trans4hexadienoylcoa and 5phenyltrans2,trans4pentadienoylcoa were determined. Such transacting ers have been reported across a variety of other fungal pks enzymes as a strategy in nature to diversify polyketides. Stereochemistry subtle differences in spatial arrangements. Peter r donald, helen mcilleron, in tuberculosis, 2009. Many workers have turned to a simple dashed line instead. Lovastatin is an important statin prescribed for the treatment and prevention of cardiovascular diseases.
Recent studies suggest that enr is also the target for the broadspectrum biocide triclosan. 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. Enoyl reductase enr, an enzyme involved in fatty acid biosynthesis, is the target for antibacterial diazaborines and the frontline antituberculosis drug isoniazid. The stereochemical course of the enoyl reduction catalyzed by fatty acid synthetase was investigated using the enzymes from rat liver and brevibacterium ammoniagenes. Conformational changes caused by inhibition of inha, the enoyl reductase from mycobacterium tuberculosis. 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.
Mechanisms of isoniazid resistance in mycobacterium. The mechanism of dienoylcoa reduction by 2,4dienoylcoa. Erb a,1, volker brecht b, georg fuchs a, michael mu. The synthesis of odd numbered fatty acids involve the use of the same extender unit, malonyl sacp, with an odd numbered starter unit. In fabi the triad is comprised of tyr 146, tyr 156, and lys 163, whereas in inha, the m. However, unlike its herbicide structural analogs, the mode of action of cyperin is not light dependent, causing loss of membrane integrity in the dark. This may be useful for protein engineering of enoyl reductases and their application in biocatalysis. Pdf chemo and stereoselective reductions are important reactions in chemistry. Biochemical and structural basis of triclosan resistance. Pdf conformational changes in 2 trans enoylacp coa. Cyperin is a natural diphenyl ether phytotoxin produced by several fungal plant pathogens. 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. Oct 28, 2019 enoyl acyl carrier protein reductase enr catalyzes the last reduction step in the bacterial type ii fatty acid biosynthesis cycle.
Enoylacyl carrier protein reductase enr catalyzes the nadhdependent stereospecific reduction of. The er shows broad substrate selectivity, reducing enoyl species including both. The stereochemistry of ketoreduction sciencedirect. 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. Inhibitor binding studies on enoyl reductase reveal conformational changes related to substrate recognition. Characterization of pseudomonas aeruginosa enoylacyl. 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. Inha has been shown to be the primary target of isoniazid. Mycobacterium tuberculosis enoylacyl carrier protein. Steadystate kinetic parameters for trans2,trans4hexadienoylcoa and 5phenyltrans2,trans4pentadienoylcoa were determined and. Enoylacylcarrierprotein reductase nadph, aspecific.
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. 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. 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. 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. Structural basis for cyclopropanation by a unique enoylacyl carrier protein reductase dheeraj khare,1 wendi a. Acyl carrier protein reductase fabi proposes a putative inhibitor binding pocket. Spectrum antibacterial drug seol 2019 bulletin of the korean chemical society wiley online library. The enoylacylcarrierprotein reductases fabi and fabl. Pdf carboxylation mechanism and stereochemistry of crotonyl. Inactivation of the inhaencoded fatty acid synthase ii.
This enzyme catalyzes the nadphdependent reduction of 2,4dienoylcoenzyme a coa thiolesters to the resulting trans3 enoyl coa. Stereochemical considerations are important in both isomerism and studies of the mechanisms of chemical reactions. Carboxylation mechanism and stereochemistry of crotonylcoa. 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 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.
Structural basis and mechanism of enoyl reductase inhibition. Substrate selectivity of an isolated enoyl reductase. A frequently occurring module catalyzes the incorporation of propionate, and reduction of the bketone group to an alcohol. 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. The chemical mechanism of the 2,4dienoylcoa reductase ec 1. Stereochemical course of enoyl reduction catalyzed by. Characterization of proteinligand binding interactions of. This transfer represents the first exception to the observation that enoyl. 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 enoyl thioester reductase inha catalyzes an essential step in fatty acid. Substrate selectivity of an isolated enoyl reductase catalytic domain.
However, inh treatment of mycobacterium tuberculosis causes the accumulation of hexacosanoic acid c 26. Functional analysis of the fusarielin biosynthetic gene cluster. Mutants in the structural gene of the inhaencoded nadhdependent 2trans enoyl acyl carrier protein reductase were identified from isoniazidresistant clinical isolates of mycobacterium tuberculosis. Biosynthesis of lovastatin uses an iterative type i polyketide synthase pks. A conserved threonine prevents selfintoxication of enoyl. The tubes containing high enzymatic activities were combined, the concentration of phosphate was adjusted to 0.
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. Acyl carrier protein reductase fabi from acinetobacter baumannii as a target for broad. Inha, the enoylthioester reductase from mycobacterium tuberculosis. This may be useful for protein engineering of enoyl. Carboxylation mechanism and stereochemistry of crotonylcoa carboxylasereductase article pdf available in proceedings of the national academy of sciences 10622. Enoylacp reductase definition of enoylacp reductase by. Structure article structural basis for cyclopropanation by a unique enoyl acyl carrier protein reductase dheeraj khare,1 wendi a. Common themes in redox chemistry emerge from the xray. The enoyl acyl carrier protein acp reductase catalyses the last step in the elongation cycle of fatty acids.
A mechanism of drug action revealed by structural studies. Carboxylation mechanism and stereochemistry of crotonyl. An entire pks consists of a series of modules that are housed within large multienzyme polypeptides. Hale,2,7 ashootosh tripathi,1 liangcai gu,1,3,8 david h. Pdf inhibitor binding studies on enoyl reductase reveal. This may be useful for protein engineering of enoyl reductases and their. A pathogenic fungi diphenyl ether phytotoxin targets plant. Escherichia colihas a single enr encoded by the essential fabigene 7, 12. Lovc is a transacting enoyl reductase er that specifically reduces three out of eight possible polyketide intermediates during lovastatin biosynthesis. Crystal structures and kinetic properties of enoylacyl.
Directed mutagenesis alters the stereochemistry of. Transfer of acetylcoa from mitochondria to cytosol. 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. The two functional enoylacyl carrier protein reductases. A cis acting enoyl reductase er catalytic domain was isolated from a. Chapterii towards the synthesis of the macrocyclic core of pladienolides a and b 41 understood way. Linking diversity in evolutionary origin and stereospecificity for enoyl thioester reductases. 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. Pdf files are great for protecting the integrity of a document, but they can be a hassle when it comes time to print antimicobcterianos. Recombinant inha proteins with defined single amino acid replacements were expressed in escherichia coli and purified to homogeneity.
It is not only a key enzyme in the pathway, but also the most diverse. The reaction was shown to proceed with transfer of the hydrogen from the pro4s position of nadph to the re face of the. In this work, multicopy plasmids expressing either inha or kasa genes were transformed into m. The assay was initiated by the addition of crotonylcoa. We report that expression of either of the two proteins restores growth of an escherichia. Transfats are associated with increased risk of cardiovascular disease and are targeted for removal from the food supply.
At high concentrations, this metabolite inhibits protoporphyrinogen oxidase, a key enzyme in porphyrin synthesis. 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. Biosyntheses of antibiotic a26771b by penicillium turbatum. The deuteriumlabeled fatty acids thus produced were subjected to.
Pkss results in the inversion of alcohol stereochemistry. Pdf carboxylation mechanism and stereochemistry of. Two genes, love and lovh, have homology with transcription factors. Prediction and manipulation of the stereochemistry of. Journal of the american chemical society 1997, 119 , 29732979. Inh is a prodrug which is biologically activated by the mtb catalaseperoxidase. Further considerations on the reaction mechanism indicated that crotonylcoa carboxylasereductase may have evolved from enoylcoa reductases. Enoyl acyl carrier protein enoyl acp reductase catalyzes the last step of the elongation cycle in the synthesis of bacterial fatty acids. The stereochemistry of complex polyketide biosynthesis citeseerx. Despite the large number of reports describing the biochemistry of this enzyme, there have been no studies that. 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. Smith1,5 1life sciences institute, university of michigan, 210 washtenaw avenue, ann arbor, mi 48109, usa. Structural basis for cyclopropanation by a unique enoyl.
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