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Charles O. Rock - One of the best experts on this subject based on the ideXlab platform.
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the structure of 3r hydroxyacyl acyl carrier protein Dehydratase fabz from pseudomonas aeruginosa
Journal of Biological Chemistry, 2004Co-Authors: Matthew S Kimber, Fernando Martin, Simon Houston, Masoud Vedadi, Akil Dharamsi, Klaus M Fiebig, Molly Schmid, Charles O. RockAbstract:Type II fatty acid biosynthesis systems are essential for membrane formation in bacteria, making the constituent proteins of this pathway attractive targets for antibacterial drug discovery. The third step in the elongation cycle of the type II fatty acid biosynthesis is catalyzed by beta-hydroxyacyl-(acyl carrier protein) (ACP) Dehydratase. There are two isoforms. FabZ, which catalyzes the dehydration of (3R)-hydroxyacyl-ACP to trans-2-acyl-ACP, is a universally expressed component of the bacterial type II system. FabA, the second isoform, as has more limited distribution in nature and, in addition to dehydration, also carries out the isomerization of trans-2- to cis-3-decenoyl-ACP as an essential step in unsaturated fatty acid biosynthesis. We report the structure of FabZ from the important human pathogen Pseudomonas aeruginosa at 2.5 A of resolution. PaFabZ is a hexamer (trimer of dimers) with the His/Glu catalytic dyad located within a deep, narrow tunnel formed at the dimer interface. Site-directed mutagenesis experiments showed that the obvious differences in the active site residues that distinguish the FabA and FabZ subfamilies of Dehydratases do not account for the unique ability of FabA to catalyze isomerization. Because the catalytic machinery of the two enzymes is practically indistinguishable, the structural differences observed in the shape of the substrate binding channels of FabA and FabZ lead us to hypothesize that the different shapes of the tunnels control the conformation and positioning of the bound substrate, allowing FabA, but not FabZ, to catalyze the isomerization reaction.
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the structure of 3r hydroxyacyl acyl carrier protein Dehydratase fabz from pseudomonas aeruginosa
Journal of Biological Chemistry, 2004Co-Authors: Matthew S Kimber, Fernando Martin, Simon Houston, Masoud Vedadi, Akil Dharamsi, Klaus M Fiebig, Molly B Schmid, Charles O. RockAbstract:Type II fatty acid biosynthesis systems are essential for membrane formation in bacteria, making the constituent proteins of this pathway attractive targets for antibacterial drug discovery. The third step in the elongation cycle of the type II fatty acid biosynthesis is catalyzed by β-hydroxyacyl-(acyl carrier protein) (ACP) Dehydratase. There are two isoforms. FabZ, which catalyzes the dehydration of (3R)-hydroxyacyl-ACP to trans-2-acyl-ACP, is a universally expressed component of the bacterial type II system. FabA, the second isoform, as has more limited distribution in nature and, in addition to dehydration, also carries out the isomerization of trans-2- to cis-3-decenoyl-ACP as an essential step in unsaturated fatty acid biosynthesis. We report the structure of FabZ from the important human pathogen Pseudomonas aeruginosa at 2.5 A of resolution. PaFabZ is a hexamer (trimer of dimers) with the His/Glu catalytic dyad located within a deep, narrow tunnel formed at the dimer interface. Site-directed mutagenesis experiments showed that the obvious differences in the active site residues that distinguish the FabA and FabZ subfamilies of Dehydratases do not account for the unique ability of FabA to catalyze isomerization. Because the catalytic machinery of the two enzymes is practically indistinguishable, the structural differences observed in the shape of the substrate binding channels of FabA and FabZ lead us to hypothesize that the different shapes of the tunnels control the conformation and positioning of the bound substrate, allowing FabA, but not FabZ, to catalyze the isomerization reaction.
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roles of the faba and fabz beta hydroxyacyl acyl carrier protein Dehydratases in escherichia coli fatty acid biosynthesis
Journal of Biological Chemistry, 1996Co-Authors: Richard J Heath, Charles O. RockAbstract:Abstract There are two genes, fabA and fabZ, encoding β-hydroxyacyl-acyl carrier protein (ACP) Dehydratases that function in the dissociated, type II fatty acid synthase system of Escherichia coli. We have investigated their roles in fatty acid synthesis by purifying the two proteins and reconstituting cycles of fatty acid synthesis in vitro using five other purified proteins. FabA and FabZ exhibited broad, overlapping chain length specificities. The FabZ Dehydratase efficiently catalyzed the dehydration of short chain β-hydroxyacyl-ACPs and long chain saturated and unsaturated β-hydroxyacyl-ACPs. FabA was most active on intermediate chain length β-hydroxyacyl-ACPs and also possessed significant activity toward both short and long chain saturated β-hydroxyacyl-ACPs. Significantly, FabA was virtually inactive in the dehydration of long chain unsaturated β-hydroxyacyl-ACP. The introduction of the double bond at the 10-carbon stage of fatty acid synthesis by FabA was only detected in the presence of β-ketoacyl-ACP synthase I (FabB). A yeast two-hybrid analysis failed to detect an interaction between FabA and FabB, therefore the channeling of intermediates toward unsaturated fatty acid synthesis by FabB was attributed to the affinity of the condensing enzyme for cis-decenoyl-ACP. The broad substrate specificity of FabZ coupled with the inactivity of FabA toward a long chain unsaturated β-hydroxyacyl-ACP provides a biochemical explanation for the phenotypes of cells with genetically altered levels of the two Dehydratases.
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roles of the faba and fabz β hydroxyacyl acyl carrier protein Dehydratases in escherichia coli fatty acid biosynthesis
Journal of Biological Chemistry, 1996Co-Authors: Richard J Heath, Charles O. RockAbstract:There are two genes, fabA and fabZ, encoding β-hydroxyacyl-acyl carrier protein (ACP) Dehydratases that function in the dissociated, type II fatty acid synthase system of Escherichia coli. We have investigated their roles in fatty acid synthesis by purifying the two proteins and reconstituting cycles of fatty acid synthesis in vitro using five other purified proteins. FabA and FabZ exhibited broad, overlapping chain length specificities. The FabZ Dehydratase efficiently catalyzed the dehydration of short chain β-hydroxyacyl-ACPs and long chain saturated and unsaturated β-hydroxyacyl-ACPs. FabA was most active on intermediate chain length β-hydroxyacyl-ACPs and also possessed significant activity toward both short and long chain saturated β-hydroxyacyl-ACPs. Significantly, FabA was virtually inactive in the dehydration of long chain unsaturated β-hydroxyacyl-ACP. The introduction of the double bond at the 10-carbon stage of fatty acid synthesis by FabA was only detected in the presence of β-ketoacyl-ACP synthase I (FabB). A yeast two-hybrid analysis failed to detect an interaction between FabA and FabB, therefore the channeling of intermediates toward unsaturated fatty acid synthesis by FabB was attributed to the affinity of the condensing enzyme for cis-decenoyl-ACP. The broad substrate specificity of FabZ coupled with the inactivity of FabA toward a long chain unsaturated β-hydroxyacyl-ACP provides a biochemical explanation for the phenotypes of cells with genetically altered levels of the two Dehydratases.
Wolfgang Buckel - One of the best experts on this subject based on the ideXlab platform.
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Phenylalanine catabolism in Archaeoglobus fulgidus VC-16
Archives of Microbiology, 2013Co-Authors: Anutthaman Parthasarathy, Jörg Kahnt, Nilanjan Pal Chowdhury, Wolfgang BuckelAbstract:Evidence is presented for a pathway of phenylalanine catabolism in the hyperthermophilic archaeon Archaeoglobus fulgidus involving the following enzymes—phenylalanine:2-oxoglutarate aminotransferase, phenyllactate dehydrogenase, radical iron–sulphur 3-phenyllactyl-CoA Dehydratase, phenylpropionyl-CoA dehydrogenase, aryl pyruvate ferredoxin oxidoreductase, ADP-forming acetyl-CoA synthetase and family III CoA-transferase. Hitherto amino acid degradation pathways involving radical iron–sulphur Dehydratases have been characterised only in mesophilic clostridia and related bacteria. The difference here is that the pathway is not fermentative but coupled to sulphate reduction. Initial experiments also show the utilisation of tryptophan as a growth substrate and the decarboxylation of caffeate by cell extracts, suggesting the potential to catabolise different classes of aromatic compounds.
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an allylic ketyl radical intermediate in clostridial amino acid fermentation
Nature, 2008Co-Authors: Daniel J Darley, Wolfgang Buckel, Antonio J PierikAbstract:In the human gut the bacterium Clostridium difficile, a common cause of hospital acquired infection worldwide, utilizes L-leucine as both oxidant and reductant. The fermentation involves a chemically demanding dehydration, catalysed by an iron–sulphur cluster-containing Dehydratase, and thought to involve ketyl radicals. This suspicion has been confirmed with the identification of a product-related allylic ketyl radical bound to the Dehydratase. The radical enzymes described previously require radical generators such as coenzyme B12, S-adenosylmethionine or oxygen. Such assistance is not necessary for the C. difficile 2-hydroxyacyl-CoA Dehydratase, making this enzyme unprecedented in biochemistry. Similar enzymes might be found in other bacteria growing anaerobically. The pathogenic bacterium Clostridium difficile thrives by fermentation of L-leucine to ammonia, CO2, isovalerate, and isocaproate under anaerobic conditions. Ketyl radicals have been proposed to mediate a key reaction catalysed by an iron–sulphur cluster-containing Dehydratase, which requires activation by ATP-dependent electron transfer from a second iron–sulphur protein. A kinetically competent product-related allylic ketyl radical bound to the Dehydratase using electron paramagnetic resonance spectroscopy is identified. These results suggest that other 2-hydroxyacyl-CoA Dehydratases (and the related benzoyl-CoA reductases) may employ ketyl radical intermediates. The absence of radical generators makes these enzymes unprecedented in biochemistry. The human pathogenic bacterium Clostridium difficile thrives by the fermentation of l-leucine to ammonia, CO2, 3-methylbutanoate and 4-methylpentanoate under anaerobic conditions1. The reductive branch to 4-methylpentanoate proceeds by means of the dehydration of (R)-2-hydroxy-4-methylpentanoyl-CoA to 4-methylpent-2-enoyl-CoA, which is chemically the most demanding step. Ketyl radicals have been proposed2 to mediate this reaction catalysed by an iron–sulphur-cluster-containing Dehydratase, which requires activation by ATP-dependent electron transfer from a second iron–sulphur protein functionally similar to the iron protein of nitrogenase. Here we identify a kinetically competent product-related allylic ketyl radical bound to the enzyme by electron paramagnetic resonance spectroscopy employing isotope-labelled (R)-2-hydroxy-4-methylpentanoyl-CoA species. We also found that the enzyme generated the stabilized pentadienoyl ketyl radical from the substrate analogue 2-hydroxypent-4-enoyl-CoA, supporting the proposed mechanism. Our results imply that also other 2-hydroxyacyl-CoA Dehydratases3 and the related benzoyl-CoA reductases4—present in anaerobically living bacteria—employ ketyl radical intermediates. The absence of radical generators such as coenzyme B12, S-adenosylmethionine or oxygen makes these enzymes unprecedented in biochemistry.
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radical enzymes in anaerobes
Annual Review of Microbiology, 2006Co-Authors: Wolfgang Buckel, Bernard T GoldingAbstract:AbstractThis review describes enzymes that contain radicals and/or catalyze reactions with radical intermediates. Because radicals irreversibly react with dioxygen, most of these enzymes occur in anaerobic bacteria and archaea. Exceptions are the families of coenzyme B12- and S-adenosylmethionine (SAM)-dependent radical enzymes, of which some members also occur in aerobes. Especially oxygen-sensitive radical enzymes are the glycyl radical enzymes and 2-hydroxyacyl-CoA Dehydratases. The latter are activated by an ATP-dependent one-electron transfer and act via a ketyl radical anion mechanism. Related enzymes are the ATP-dependent benzoyl-CoA reductase and the ATP-independent 4-hydroxybenzoyl-CoA reductase. Ketyl radical anions may also be generated by one-electron oxidation as shown by the flavin-adenine-dinucleotide (FAD)- and [4Fe-4S]-containing 4-hydroxybutyryl-CoA Dehydratase. Finally, two radical enzymes are discussed, pyruvate:ferredoxin oxidoreductase and methane-forming methyl-CoM reductase, which ...
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crystal structure of 4 hydroxybutyryl coa Dehydratase radical catalysis involving a 4fe 4s cluster and flavin
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Berta M Martins, Wolfgang Buckel, Holger Dobbek, Irfan Cinkaya, Albrecht MesserschmidtAbstract:Dehydratases catalyze the breakage of a carbon—oxygen bond leading to unsaturated products via the elimination of water. The 1.6-A resolution crystal structure of 4-hydroxybutyryl-CoA Dehydratase from the γ-aminobutyrate-fermenting Clostridium aminobutyricum represents a new class of Dehydratases with an unprecedented active site architecture. A [4Fe–4S]2+ cluster, coordinated by three cysteine and one histidine residues, is located 7 A from the Re-side of a flavin adenine dinucleotide (FAD) moiety. The structure provides insight into the function of these ubiquitous prosthetic groups in the chemically nonfacile, radical-mediated dehydration of 4-hydroxybutyryl-CoA. The substrate can be bound between the [4Fe–4S]2+ cluster and the FAD with both cofactors contributing to its radical activation and catalytic conversion. Our results raise interesting questions regarding the mechanism of acyl-CoA dehydrogenases, which are involved in fatty acid oxidation, and address the divergent evolution of the ancestral common gene.
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molecular characterization of phenyllactate Dehydratase and its initiator from clostridium sporogenes
Molecular Microbiology, 2002Co-Authors: Sandra Dickert, Antonio J Pierik, Wolfgang BuckelAbstract:The heterotrimeric phenyllactate Dehydratase from Clostridium sporogenes, FldABC, catalyses the reversible dehydration of (R)-phenyllactate to (E)-cinnamate in two steps: (i) CoA-transfer from the cofactor cinnamoyl-CoA to phenyllactate to yield phenyllactyl-CoA and the product cinnamate mediated by FldA, a (R)-phenyllactate CoA-transferase; followed by (ii) dehydration of phenyllactyl-CoA to cinnamoyl-CoA mediated by heterodimeric FldBC, a phenyllactyl-CoA Dehydratase. Phenyllactate Dehydratase requires initiation by ATP, MgCl 2 and a reducing agent such as dithionite mediated by an extremely oxygen-sensitive initiator protein (FldI) present In the cell-free extract. All four genes coding for these proteins were cloned and shown to be clustered in the order fldAIBC, which shares over 95% sequence identity of nucleotide and protein levels with a gene cluster detected in the genome of the closely related Clostridium botulinum Hall strain A. FldA shows sequence similarities to a new family of CoA-transferases, which apparently do not form covalent enzyme CoA-ester intermediates. An N-terminal Strep II-Tag containing enzymatically active FldI was overproduced and purified from Escherichia coli. FldI was characterized as a homodimeric protein, which contains one [4Fe-4S] 1 + / 2 + cluster with an electron spin S= 3/2 in the reduced form. The amino acid sequence as well as the chemical and EPR-properties of the pure protein are very similar to those of component A of 2-hydroxyglutaryl-CoA Dehydratase from Acidaminococcus fermentans (HgdC), which was able to replace FldI in the activation of phenyllactate Dehydratase. Only in the oxidized state, FldI and component A exhibit significant ATPase activity, which appears to be essential for unidirectional electron transfer. Both subunits of phenyllactyl-CoA Dehydratase (FldBC) show significant sequence similarities to both subunits of 2-hydroxyglutaryl-CoA Dehydratase (HgdAB). The fldAIBC gene cluster resembles the hadAIBC gene cluster in the genome of Clostridium difficile and the hadABC,I genes in C. botulinum. The four subunits of these deduced 2-hydroxyacid Dehydratases (65-81% amino acid sequence identity between the had genes) probably code for a 2-hydroxyisocaproate Dehydratase involved in leucine fermentation. This enzyme could be the target for metronidazole in the treatment of pseudomembranous enterocolitis caused by C. difficile.
Nawel Slama - One of the best experts on this subject based on the ideXlab platform.
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Discovery of a novel Dehydratase of the fatty acid synthase type II critical for ketomycolic acid biosynthesis and virulence of Mycobacterium tuberculosis
Scientific Reports, 2020Co-Authors: Cyril Lefebvre, Wafa Frigui, Françoise Lauzeral-vizcaino, Patricia Constant, Tanya Parish, Nawel Slama, Anne Lemassu, Nathalie Eynard, Mamadou DafféAbstract:The fatty acid synthase type II (FAS-II) multienzyme system builds the main chain of mycolic acids (MAs), important lipid pathogenicity factors of Mycobacterium tuberculosis (Mtb). Due to their original structure, the identification of the (3 R)-hydroxyacyl-ACP Dehydratases, HadAB and HadBC, of Mtb FAS-II complex required in-depth work. Here, we report the discovery of a third Dehydratase protein, HadDMtb (Rv0504c), whose gene is non-essential and sits upstream of cmaA2 encoding a cyclopropane synthase dedicated to keto- and methoxy-MAs. HadDMtb deletion triggered a marked change in Mtb keto-MA content and size distribution, deeply impacting the production of full-size molecules. Furthermore, abnormal MAs, likely generated from 3-hydroxylated intermediates, accumulated. These data strongly suggest that HadDMtb catalyzes the 3-hydroxyacyl dehydratation step of late FAS-II elongation cycles during keto-MA biosynthesis. Phenotyping of Mtb hadD deletion mutant revealed the influence of HadDMtb on the planktonic growth, colony morphology and biofilm structuration, as well as on low temperature tolerance. Importantly, HadDMtb has a strong impact on Mtb virulence in the mouse model of infection. The effects of the lack of HadDMtb observed both in vitro and in vivo designate this protein as a bona fide target for the development of novel anti-TB intervention strategies.
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a common mechanism of inhibition of the mycobacterium tuberculosis mycolic acid biosynthetic pathway by isoxyl and thiacetazone
Journal of Biological Chemistry, 2012Co-Authors: Anna E Grzegorzewicz, Nawel Slama, Jana Kordulakova, Victoria Jones, Sarah E M Born, Juan Manuel Belardinelli, Adrien Vaquie, Vijay A K B Gundi, Jan MadackiAbstract:Isoxyl (ISO) and thiacetazone (TAC), two prodrugs once used in the clinical treatment of tuberculosis, have long been thought to abolish Mycobacterium tuberculosis (M. tuberculosis) growth through the inhibition of mycolic acid biosynthesis, but their respective targets in this pathway have remained elusive. Here we show that treating M. tuberculosis with ISO or TAC results in both cases in the accumulation of 3-hydroxy C(18), C(20), and C(22) fatty acids, suggestive of an inhibition of the Dehydratase step of the fatty-acid synthase type II elongation cycle. Consistently, overexpression of the essential hadABC genes encoding the (3R)-hydroxyacyl-acyl carrier protein Dehydratases resulted in more than a 16- and 80-fold increase in the resistance of M. tuberculosis to ISO and TAC, respectively. A missense mutation in the hadA gene of spontaneous ISO- and TAC-resistant mutants was sufficient to confer upon M. tuberculosis high level resistance to both drugs. Other mutations found in hypersusceptible or resistant M. tuberculosis and Mycobacterium kansasii isolates mapped to hadC. Mutations affecting the non-essential mycolic acid methyltransferases MmaA4 and MmaA2 were also found in M. tuberculosis spontaneous ISO- and TAC-resistant mutants. That MmaA4, at least, participates in the activation of the two prodrugs as proposed earlier is not supported by our biochemical evidence. Instead and in light of the known interactions of both MmaA4 and MmaA2 with HadAB and HadBC, we propose that mutations affecting these enzymes may impact the binding of ISO and TAC to the Dehydratases.
June E Ayling - One of the best experts on this subject based on the ideXlab platform.
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can the dcohα isozyme compensate in patients with 4a hydroxy tetrahydrobiopterin Dehydratase dcoh deficiency
Molecular Genetics and Metabolism, 2006Co-Authors: Joan M Hevel, Jason A Stewart, Katherine L Gross, June E AylingAbstract:4a-Hydroxy-tetrahydrobiopterin Dehydratase/DCoH is a bifunctional protein. In the cytoplasm it is an enzyme required for the regeneration of tetrahydrobiopterin, an essential cofactor for phenylalanine hydroxylase. In the nucleus it functions as a transcriptional coactivator by forming a 2:2 heterotetramer with the hepatic nuclear factor HNF1alpha (HNF1). Patients with a deficiency of Dehydratase activity have elevated levels of phenylalanine, and accumulate 7-pterins due to degradation of its substrate 4a-hydroxy-tetrahydrobiopterin. Curiously, the hyperphenylalaninemia is transient, and no defects in the transcriptional coactivator function have been reported. Recently, a human isozyme, Dehydratase/DCoHalpha, has been detected which shares 60% identity with Dehydratase/DCoH. This investigation was undertaken to ascertain if Dehydratase/DCoHalpha has the pre-requisite properties to compensate in individuals lacking an active form of DCoH. DCoHalpha demonstrated the ability to quantitatively alter HNF1-dependent DNA-binding in vitro whereas DCoH was ineffective in vitro. This characteristic, due to the presence of dimeric DCoHalpha, demonstrates that DCoHalpha does not require any additional mammalian regulation process to alter DNA binding and therefore, may be more effective than DCoH at low concentrations. The Dehydratase activity of each isoform was measured by a direct spectrophotometric assay. Km and Vmax for DCoHalpha were both 2-3 times higher than for DCoH, thus leaving the catalytic efficiency (Vmax/Km) the same for both enzymes. In conclusion, the properties of Dehydratase/DCoHalpha are consistent with the hypothesis that the activity of this isozyme could account for the relatively mild symptoms reported for patients with a defect in Dehydratase/DCoH.
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hyperphenylalaninemia and 7 pterin excretion associated with mutations in 4a hydroxy tetrahydrobiopterin Dehydratase dcoh analysis of enzyme activity in intestinal biopsies
Molecular Genetics and Metabolism, 2000Co-Authors: June E Ayling, Steven W Bailey, Scott R Boerth, R Giugliani, Christian Braegger, Beat Thony, Nenad BlauAbstract:Abstract Hyperphenylalaninemia, which can cause neurological disorders and mental retardation, results from a mutation in phenylalanine hydroxylase or an enzyme required for biosynthesis or regeneration of its cofactor, tetrahydrobiopterin. The hyperphenylalaninemia variant primapterinuria is characterized by the excretion of 7-biopterin (primapterin). This disorder is thought to be due to a deficiency of 4a-hydroxy-tetrahydrobiopterin Dehydratase (pterin-4a-carbinolamine Dehydratase), but a lack of tissue activity has not been directly demonstrated. The five mutations so far recognized in patients with primapterinuria are associated with either a single amino acid change or a premature stop codon. Only C81R has been successfully expressed in soluble form, and was found to have 40% of normal activity. Tissues which could be obtained by minimally invasive procedures were analyzed for Dehydratase activity. None was detected in normal human white cells or fibroblasts. However, activity was found in intestine of rat, dog, pig, and particularly humans where it was only eight times lower than in liver. Distribution along the length and across the wall of small intestine was relatively uniform. Moreover, the Dehydratases from human liver and intestinal mucosa have identical kinetic properties. A biopsy of duodenal mucosa from a patient with homozygous E96K Dehydratase had activity of 55 nmol · min−1g−1 mucosa compared to 329 ± 32 nmol · min−1g−1 tissue in controls (n = 12). The sixfold lower tissue activity of the E96K mutant alone may not be sufficient to account for the biochemical symptoms of primapterinuria in this patient. However, accumulation of a 4a-hydroxy-tetrahydrobiopterin degradation product (a side-chain cyclic adduct), which has been observed in vitro and appears to be a Dehydratase inhibitor, may further exacerbate the problem.
Richard J Heath - One of the best experts on this subject based on the ideXlab platform.
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roles of the faba and fabz beta hydroxyacyl acyl carrier protein Dehydratases in escherichia coli fatty acid biosynthesis
Journal of Biological Chemistry, 1996Co-Authors: Richard J Heath, Charles O. RockAbstract:Abstract There are two genes, fabA and fabZ, encoding β-hydroxyacyl-acyl carrier protein (ACP) Dehydratases that function in the dissociated, type II fatty acid synthase system of Escherichia coli. We have investigated their roles in fatty acid synthesis by purifying the two proteins and reconstituting cycles of fatty acid synthesis in vitro using five other purified proteins. FabA and FabZ exhibited broad, overlapping chain length specificities. The FabZ Dehydratase efficiently catalyzed the dehydration of short chain β-hydroxyacyl-ACPs and long chain saturated and unsaturated β-hydroxyacyl-ACPs. FabA was most active on intermediate chain length β-hydroxyacyl-ACPs and also possessed significant activity toward both short and long chain saturated β-hydroxyacyl-ACPs. Significantly, FabA was virtually inactive in the dehydration of long chain unsaturated β-hydroxyacyl-ACP. The introduction of the double bond at the 10-carbon stage of fatty acid synthesis by FabA was only detected in the presence of β-ketoacyl-ACP synthase I (FabB). A yeast two-hybrid analysis failed to detect an interaction between FabA and FabB, therefore the channeling of intermediates toward unsaturated fatty acid synthesis by FabB was attributed to the affinity of the condensing enzyme for cis-decenoyl-ACP. The broad substrate specificity of FabZ coupled with the inactivity of FabA toward a long chain unsaturated β-hydroxyacyl-ACP provides a biochemical explanation for the phenotypes of cells with genetically altered levels of the two Dehydratases.
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roles of the faba and fabz β hydroxyacyl acyl carrier protein Dehydratases in escherichia coli fatty acid biosynthesis
Journal of Biological Chemistry, 1996Co-Authors: Richard J Heath, Charles O. RockAbstract:There are two genes, fabA and fabZ, encoding β-hydroxyacyl-acyl carrier protein (ACP) Dehydratases that function in the dissociated, type II fatty acid synthase system of Escherichia coli. We have investigated their roles in fatty acid synthesis by purifying the two proteins and reconstituting cycles of fatty acid synthesis in vitro using five other purified proteins. FabA and FabZ exhibited broad, overlapping chain length specificities. The FabZ Dehydratase efficiently catalyzed the dehydration of short chain β-hydroxyacyl-ACPs and long chain saturated and unsaturated β-hydroxyacyl-ACPs. FabA was most active on intermediate chain length β-hydroxyacyl-ACPs and also possessed significant activity toward both short and long chain saturated β-hydroxyacyl-ACPs. Significantly, FabA was virtually inactive in the dehydration of long chain unsaturated β-hydroxyacyl-ACP. The introduction of the double bond at the 10-carbon stage of fatty acid synthesis by FabA was only detected in the presence of β-ketoacyl-ACP synthase I (FabB). A yeast two-hybrid analysis failed to detect an interaction between FabA and FabB, therefore the channeling of intermediates toward unsaturated fatty acid synthesis by FabB was attributed to the affinity of the condensing enzyme for cis-decenoyl-ACP. The broad substrate specificity of FabZ coupled with the inactivity of FabA toward a long chain unsaturated β-hydroxyacyl-ACP provides a biochemical explanation for the phenotypes of cells with genetically altered levels of the two Dehydratases.