Β-hydroxydecanoyl Thioester Dehydrase Research Articles

Expression of theEscherichia coli fabA gene encoding β-hydroxydecanoyl thioester dehydrase and transport to chloroplasts in transgenic tobacco

The fabA gene of Escherichia coli encodes beta-hydroxydecanoyl thioester dehydrase (HDDase), a pivotal enzyme in the biosynthesis of the unsaturated fatty acid cis-vaccenic acid, through the anaerobic pathway. This enzyme is specific to bacterial fatty acid biosynthetic pathways, although other enzymes for fatty acid synthesis are very similar in plants and bacteria. We constructed chimaeric plant expression vectors, pfab21 and pfab22, carrying the fabA gene under the transcriptional control of the cauliflower mosaic virus (CaMV) promoter of 35S RNA. In pfab21, fabA was placed directly under the control of the CaMV 35S promoter; whereas in pfab22, the DNA sequence coding for the chloroplast-targeting transit peptide (TP) of the pea ribulose-1,5-bisphosphate carboxylase (RuBisCo) small subunit was fused to the fabA gene in order to allow transport of HDDase to the chloroplast, the organelle responsible for de novo fatty acid biosynthesis in plants. Transgenic plants of Nicotiana tabacum were obtained by Agrobacterium-mediated transformation with pfab21 or pfab22. Expression of fabA transcripts of sizes expected from the chimaeric constructs was shown by RNA blot hybridization. The HDDase protein derived from pfab22 was correctly processed and transported to chloroplasts in transformed plants. The enzymatic activity of HDDase was also detected in chloroplasts isolated from the transformants derived from pfab22 (but not pfab21) and in total leaf protein of all transformants. However, no significant changes were observed in the fatty acid compositions, including cis-vaccenic acid, of leaf chloroplasts and self-fertilized seeds. These results are discussed in relation with the possible structural organization of plant fatty acid synthase.

Escherichia coli β-hydroxydecanoyl thioester dehydrase reacts with native C 10 acyl-acyl-carrier proteins of plant and bacterial origin

β — Hydroxydecanoyl — [acyl — carrier — protein] dehydrase catalyzes the essential step in the formation of unsaturated fatty acids in Escherichia coli. This reaction was characterized with native C 10 acyl—acyl-carrier protein (ACP) structures in both an aqueous phase system and a substrate immobilization assay system. The dehydrase is equally active with E. coli ACP, recombinant ACP-I derived from spinach, or protein A:ACP-I fusion (acyl-thioesters). There were differences among the substrates in terms of the equilibrium product distribution. Both E. coli acyl-ACP and recombinant acyl-ACP-I as cosubstrates with β-OH 10:0, trans-2 10:1, or cis-3 10:1 yielded about equal amounts (37 mol%) of the two monoenes regardless of the initial substrate. In contrast, the fusion acyl-ACP-I yielded only 17 mol% cis-3 10:1 with 49 mol% trans-2 10:1 present at equilibrium. These equilibrium values for native cis-3 10:1 are higher than those reported previously for the dehydrase using N-acetylcysteamine thioesters as substrates. The K m values for each β-OH 10:0 ACP substrate were similar to each other and within the range of in vivo concentrations (5–10 μ m). Dehydrase reactivity depends more on acyl chain length than ACP structure or origin and is therefore different from other branch point ACP-utilizing enzymes (plant and bacterial) which discriminate according to ACP structure (D. J. Guerra, J. B. Ohlrogge, and M. Frentzen, 1986, Plant Physiol. 82, 448–453).

The beginnings of enzyme suicide

TheE. Coli enzyme β-hydroxydecanoyl thioester dehydrase catalyzes the conversion of 3-hydroxydecanoyl-thioester to the corresponding 2,3 enoate and also the reversible isomerization of 2,3- and 3,4 enoates, 3-decynoyl thioesters inhibit all these reactions irreversibly. The mechanism responsible for these inhibitions involves enzyme-catalyzed conversion of 3-decynoyl thioester to 2,4 decadienoyl thioester followed by covalent interaction of the allene with an active site histidine. Enzyme inactivation results from the ability of the enzyme to convert a substrate analogue into a potent active site modifying reagent. A brief review is given of research on unsaturated fatty acid synthesis which inadvertently revealed the phenomenon of “Enzyme Suicide”.

Stereochemical course of the hydration reaction catalysed by β-hydroxydecanoylthioester dehydrase

Enzyme-catalysed hydration of the N-acetylcysteamine thioester of (E)-dec-2-enoic acid to the corresponding thioester of (R)-3-hydroxydecanoic acid proceeds in a syn fashion, with protonation on the si face at substrate C-2.

β-Hydroxydecanoylthioester dehydrase: A rapid, convenient, and accurate product distribution assay

High-performance liquid chromatography on silica gel has been used to separate the products from incubation of substrates with β-hydroxydecanoylthioester dehydrase ( Escherichia coli). Peaks are detected by their absorbances at 230 nm. Following correction for differences in extinction coefficients, comparison of the peak areas reveals the relative amounts of β-hydroxydecanoate, E-2-decenoate, and Z-3-decenoate thioesters of N-acetylcysteamine.

β-Hydroxydecanoylthioester dehydrase. Steric course at substrate C-2 and overall steric course of the enzyme–catalysed allylic rearrangement

β-Hydroxydecanoylthioester dehydrase (Escherichia coli)catalyses the addition of a proton to the si face at C-2 of the N-acetylcysteinamine thioester of E-dec-2-enoic acid in isomerzing this substance to the corresponding Z-dec-3-enoic acid thioester with the result that the overall steric course of the allylic rearrangement is suprafacial.

Novel application of affinity chromatography with chaotropic elution from Blue Dextran-Sepharose for the purification of β-hydroxydecanoyl thioester dehydrase from Escherichia coli

Novel application of affinity chromatography with chaotropic elution from Blue Dextran-Sepharose for the purification of β-hydroxydecanoyl thioester dehydrase from Escherichia coli

One-step gene amplification by Mu-mediated transposition of E. coli genes to a multicopy plasmid

A general in vivo method to amplify the number of copies of a specific gene in one step is described. The method is directly applicable to any selectable gene of Escherichia coli and is based on the Mu-mediated transposition of segments of host chromosomes into the conjugative, multicopy plasmid R6K. Using this method we have cloned the β-hydroxydecanoyl thioester dehydrase structural gene, fabA, into the R6K plasmid. Strains carrying the resultant plasmid produced 13 to 21 times more dehydrase than control strains.

Inhibition of [formula omitted] thioester dehydrase by ppGpp

Guanosine-5′-diphosphate-3′-diphosphate was found to inhibit β-hydroxydecanoyl thioester dehydrase of E. coli at concentrations corresponding to those generated in vivo during amino acid starvation of rel + cells.

Synthesis of Unsaturated Fatty Acids and the Lesion in fab B Mutants

Two classes of Escherichia coli unsaturated fatty acid auxotrophs, which are complementary both in vivo and in vitro, have been utilized to study unsaturated fatty acid synthesis. β-Hydroxydecanoyl thioester dehydrase, which catalyzes the first committed reaction in the unsaturated fatty acid synthetic pathway, is defective in one class, fab A. The defect in the second class, fab B, was unknown. The unsaturated fatty acid synthetic activity defective in fab B extracts was isolated from fab A extracts on the basis of an in vitro complementation assay. Throughout a 586-fold purification procedure the fab A unsaturated fatty acid synthetic activity was associated with β-ketoacyl acyl carrier protein (ACP) synthetase, suggesting that the two activities are catalyzed by the same protein. Further support for this suggestion derived from the findings that both activities were purified together during the isolation of homogeneous β-ketoacyl-ACP synthetase from wild type E. coli B, both were inhibited to the same extent by iodoacetamide, both were protected against iodoacetamide inhibition by acetyl-ACP, and both were inactivated at 43° at identical rates. The fact that wild type β-ketoacyl-ACP synthetase stimulated unsaturated fatty acid synthesis by fab B extracts suggested that fab B β-ketoacyl-ACP synthetase is defective in unsaturated fatty acid synthetic activity. Although partially purified fab B β-ketoacyl-ACP synthetase was shown to be defective in unsaturated fatty acid synthetic activity, the mutant enzyme catalyzed normally condensation reactions involving fatty acyl-ACPs that are intermediates in the synthesis of saturated and unsaturated fatty acids. Although the unsaturated fatty acid synthetic activity of β-ketoacyl-ACP synthetase is not understood, these studies demonstrate that this enzyme catalyzes a unique reaction in the synthesis of unsaturated fatty acids.

An Estimate of the Minimum Amount of Unsaturated Fatty Acid Required for Growth of Escherichia coli

Abstract A temperature-sensitive unsaturated fatty acid auxotroph was used to demonstrate that the fabA gene of Escherichia coli is the structual gene for the β-hydroxydecanoyl thioester dehydrase studied by Bloch and co-workers. This mutant, which requires an exogenous source of unsaturated fatty acid for growth at temperatures above 36°, possesses a dehydrase activity of greatly increased thermolability. Analysis of fab+ and fab- transductants and of fab+ revertants indicate that the temperature-sensitive dehydrase activity is responsible for the growth phenotype of this strain. This mutant was then used for an estimation of the minimum amount of unsaturated fatty acid needed for growth. Due to its temperature-sensitive phenotype, at semipermissive temperatures the strain grows at a subnormal rate due to a reduced rate of unsaturated fatty acid synthesis. Under such conditions, a minimum amount of unsaturated fatty acid is reached which is 15 to 20% of the total phospholipid fatty acid. No marked accumulation of short chain saturated fatty acids is seen during growth at semipermissive temperatures. A similar study with another strain showed that the minimum content of unsaturated fatty acid needed for growth is largely independent of the ratio of palmitoleate to cis-vaccenate in the phospholipids. These results are discussed in terms of the role of unsaturated fatty acids in membrane structure and function and also in terms of the control of fatty acid synthesis.

Acyl Carrier Protein: XVII. PURIFICATION AND PROPERTIES OF β-HYDROXYACYL ACYL CARRIER PROTEIN DEHYDRASE

Abstract β-Hydroxyacyl acyl carrier protein (ACP) dehydrase has been purified 2900-fold from extracts of Escherichia coli. The enzyme catalyzes the reversible dehydration of β-hydroxyacyl-ACP thioesters to yield specifically trans-2-enoyl-ACP products. It is active with trans-2-enoyl-ACP thioesters of chain lengths from 4 through 16 carbon atoms. The enzyme catalyzes the hydration of cis-5-trans-2-dodecadienoyl-ACP, an intermediate in unsaturated fatty acid synthesis, as well as the trans-2-enoyl-ACP substrates that are intermediates in saturated fatty acid synthesis. Thus this enzyme can function in the synthesis of both saturated and unsaturated fatty acids. The lowest enzyme activity was noted with trans-2-decenoyl-ACP. The relevance of this finding with respect to the activity of β-hydroxydecanoyl thioester dehydrase, which specifically catalyzes the conversion of β-hydroxydecanoyl-ACP to cis-3-decenoyl-ACP is discussed as a possible factor in control of saturated versus unsaturated fatty acid synthesis. Attempts to separate or to show distinguishing characteristics of β-hydroxyacyl-ACP dehydrase activities with short, medium, or long chain substrates were unsuccessful, and these activities are therefore attributed to a single enzyme.

On the mode of interaction of -hydroxydecanoyl thioester dehydrase with allenic acid derivatives.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMode of interaction of β-hydroxydecanoyl thioester dehydrase with allenic acid derivativesMasuo Morisaki and Konrad BlochCite this: Biochemistry 1972, 11, 3, 309–314Publication Date (Print):February 1, 1972Publication History Published online1 May 2002Published inissue 1 February 1972https://doi.org/10.1021/bi00753a001RIGHTS & PERMISSIONSArticle Views109Altmetric-Citations37LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (712 KB) Get e-Alerts Get e-Alerts

Inhibition of β-hydroxydecanoyl thioester dehydrase by some allenic acids and their thioesters

The allenic substrate analogue, 2,3-decadienoyl- N-acetylcys teamine, and free 2,3-decadienoic acid were previously shown to inhibit β-hydroxydecanoyl thioester dehydrase irreversibly. Racemic 2,3-decadienoic acid has now been resolved via its amphetamine salts. The dextrorotatory isomer is found to be twice as potent an inhibitor as the racemic acid, while the levorotatory allene affects enzyme activity only slightly. A series of inhibitors varying in the length of the acyl chain (C 8C 14) has been prepared. Enzyme inactivation occurs most rapidly with the C 10-congener of the acetylenic and allenic thioesters, while the C 12 homologue is the most potent of the free allenic acid inhibitors.

Mode of Inhibition of β-Hydroxydecanoyl Thioester Dehydrase by 3-Decynoyl-N-acetylcysteamine

Abstract The alkynoic substrate analogue 3-decynoyl-N-acetylcysteamine (NAC) was previously shown to inhibit β-hydroxydecanoyl thioester dehydrase irreversibly. 2,3-Decadienoyl-NAC prepared by chemical isomerization of 3-decynoyl-NAC inhibits dehydrase activity even more effectively. The allenic and the acetylenic inhibitors both inactivate dehydrase by combining with a histidine residue at the active site. α-Dideutero-3-decynoyl-NAC reacts more slowly with dehydrase than its hydrogen analogue: kh:kd = 2.60. No kinetic isotope effect is observed for the inhibition of enzyme by α-d-2,3-decadienoyl-NAC. It is concluded that 3-decynoyl-NAC as such is not the true inhibitor, but that the dehydrase isomerizes it to the allene which in turn inactivates the enzyme by a chemical reaction. The finding that free 2,3-decadienoic acid inhibits dehydrase, whereas 3-decynoic acid does not, supports the postulated mode of action.

Enzyme inhibition by acetylenic compounds

Soybean lipoxidase and prostaglandin synthetase from sheep seminal vesicles are subject to powerful inhibition by 5,8,11,14-eicosatetraynoic acid, apparently by a slow but irreversible attack on the enzymes. It is postulated that inhibition may result from conversion of the acetylenic compound to a reactive allene by the enzymes studied, as has been proposed in the inhibition of β-hydroxydecanoyl thioester dehydrase by 3-decynoyl-N-acetylcysteamine.

β-Hydroxydecanoyl Thioester Dehydrase: STUDIES ON MOLECULAR STRUCTURE AND ACTIVE SITE

Abstract β-Hydroxydecanoyl thioester dehydrase from E. coli has been shown to have a molecular weight of 36,000 by ultracentrifugation, gel filtration, and amino acid composition. Electrophoretic behavior, however, suggests that the protein molecule is composed of two equal, if not identical, polypeptide chains of molecular weight 18,000. The enzyme also contains 4 half-cystinyl residues per 36,000 g of protein. A variety of chemical modifications, including photooxidation, alkylation, and nitration, lead to the conclusion that 1 histidyl and 1 tyrosyl residue are essential for the catalytic activity of the enzyme. The pH-kcat profile for the isomerization reaction is consistent with the participation of these two amino acids in the catalytic process. Evidence is presented that 3-decynoyl-N-acetylcysteamine, a specific dehydrase inhibitor, reacts covalently with the same histidyl residue and protects the enzyme against the modifying active site-directed reagents. Conversely, nitrated or alkylated enzyme has lost the ability to react with the acetylenic inhibitor. No treatment has been found which will dissociate dehydration from isomerization activity, supporting the concept that the dehydrase is a truly multifunctional enzyme.

Mechanism of Action of β-Hydroxydecanoyl Thioester Dehydrase

Abstract Studies on the mechanism of the reversible enzymatic dehydration of d(-)-β-hydroxydecanoyl-N-acetylcysteamine to trans-2-decenoyl-N-acetylcysteamine and cis-3-decanoyl-N-acetylcysteamine are presented. They include determination of kinetic isotope effects with deuterium-labeled substrates and labeling experiments designed to follow the fates of the α- and γ-hydrogen atoms during the course of the reactions. The results show that trans-2-decenoyl-N-acetylcysteamine is formed directly by dehydration of β-hydroxydecanoate, whereas cis-3-decenoyl-N-acetylcysteamine arises only by isomerization of the conjugated enoate. The present findings, in conjunction with earlier kinetic data, lead to the conclusion that interconversion of the three substrates involves a common intermediate, presumably enzyme-bound α,β-decenoate.

β-Hydroxydecanoyl Thioester Dehydrase: SPECIFICITY OF SUBSTRATES AND ACETYLENIC INHIBITORS

Abstract The enzyme β-hydroxydecanoyl thioester dehydrase, a requisite component of the fatty acid synthetase system of Escherichia coli, catalyzes the reversible interconversion of the thioesters of β-hydroxydecanoic acid, trans-α,β-decenoic acid, and cis-β, γ-decenoic acid. All activities of the dehydrase are strongly inhibited by 3-decynoyl-N-acetylcysteamine. A marked preference for C10 substrates has previously been noted. For further elucidation of dehydrase specificity, kinetic parameters for the enzymatic dehydration and isomerization were determined with a series of substrates varying in chain length from eight to 12 carbon atoms in the acyl moiety. Inhibition constants for β,γ-acetylenic thioesters of the same series were also measured. The data show a remarkable parallelism between the relative activities of substrates and inhibitors as a function of carbon chain length. The inhibitor 3-decynoyl-N-acetylcysteamine appears to be irreversibly bound to the dehydrase.

β-Hydroxydecanoyl Thioester Dehydrase: II. MODE OF ACTION

The β-hydroxydecanoyl thioester dehydrase of Escherichia coli has a high degree of chain length specificity, catalyzing the dehydration of β-hydroxydecanoyl-N-acetylcysteamine at more than 10 times the rate of the corresponding C8 and C12 compounds. The enzyme acts on different thioesters of β-hydroxydecanoic acid at the following relative rates: acyl carrier protein, 6; N-acetylcysteamine, 1; and pantetheine, 0.6. In each case, both α,β- and β,γ-unsaturated products are formed. The dehydrase also catalyzes the interconversion and hydration of the isomeric decenoyl thioesters. The same equilibrium mixture is obtained when either β-hydroxydecanoyl-, trans-α,β-decenoyl-, or cis-β,γ-decenoyl-N-acetylcysteamine is used as substrate. Experiments are described which attempt to delineate the pathway of interconversion of the different substrates, and which argue for a single, multifunctional enzyme catalyzing the transformations. In accord with the proposed pathway for the synthesis of long chain acids in E. coli, the bacterial fatty acid synthetase catalyzes the chain elongation of α,β-decenoyl acyl carrier protein to mainly saturated acids, and of β-hydroxydecanoyl and β,γ-decenoyl acyl carrier protein, to mixtures of long chain saturated and unsaturated acids. Explanations are offered for the apparent paradox that the isolated dehydrase converts β-hydroxydecanoyl thioesters primarily to α,β-decenoate, whereas the fatty acid synthetase, of which the dehydrase is a component, catalyzes the formation of predominantly unsaturated acids, the elongation products of β,γ-decenoate.