Overproduction of a Functional Fatty Acid Biosynthetic Enzyme Blocks Fatty Acid Synthesis in Escherichia coli

β-Ketoacyl-acyl carrier protein (ACP) synthetase II (KAS II) is one of three Escherichia coli isozymes that catalyze the elongation of growing fatty acid chains by condensation of acyl-ACP with malonyl-ACP. Overexpression of this enzyme has been found to be extremely toxic to E. coli, much more so than overproduction of either of the other KAS isozymes, KAS I or KAS III. The immediate effect of KAS II overproduction is the cessation of phospholipid synthesis, and this inhibition is specifically due to the blockage of fatty acid synthesis. To determine the cause of this inhibition, we examined the intracellular pools of ACP, coenzyme A (CoA), and their acyl thioesters. Although no significant changes were detected in the acyl-ACP pools, the CoA pools were dramatically altered by KAS II overproduction. Malonyl-CoA increased to about 40% of the total cellular CoA pool upon KAS II overproduction from a steady-state level of around 0.5% in the absence of KAS II overproduction. This finding indicated that the conversion of malonyl-CoA to fatty acids had been blocked and could be explained if either the conversion of malonyl-CoA to malonyl-ACP and/or the elongation reactions of fatty acid synthesis had been blocked. Overproduction of malonyl-CoA:ACP transacylase, the enzyme catalyzing the conversion of malonyl-CoA to malonyl-ACP, partially relieved the toxicity of KAS II overproduction, consistent with a model in which high levels of KAS II blocks access of the other KAS isozymes to malonyl-CoA:ACP transacylase.     

Identification and molecular characterization of the beta-ketoacyl-[acyl carrier protein] synthase component of the Arabidopsis mitochondrial fatty acid synthase

Substrate specificity of condensing enzymes is a predominant factor determining the nature of fatty acyl chains synthesized by type II fatty acid synthase (FAS) enzyme complexes composed of discrete enzymes. The gene (mtKAS) encoding the condensing enzyme, beta-ketoacyl-[acyl carrier protein] (ACP) synthase (KAS), constituent of the mitochondrial FAS was cloned from Arabidopsis thaliana, and its product was purified and characterized. The mtKAS cDNA complemented the KAS II defect in the E. coli CY244 strain mutated in both fabB and fabF encoding KAS I and KAS II, respectively, demonstrating its ability to catalyze the condensation reaction in fatty acid synthesis. In vitro assays using extracts of CY244 containing all E. coli FAS components, except that KAS I and II were replaced by mtKAS, gave C(4)-C(18) fatty acids exhibiting a bimodal distribution with peaks at C(8) and C(14)-C(16). Previously observed bimodal distributions obtained using mitochondrial extracts appear attributable to the mtKAS enzyme in the extracts. Although the mtKAS sequence is most similar to that of bacterial KAS IIs, sensitivity of mtKAS to the antibiotic cerulenin resembles that of E. coli KAS I. In the first or priming condensation reaction of de novo fatty acid synthesis, purified His-tagged mtKAS efficiently utilized malonyl-ACP, but not acetyl-CoA as primer substrate. Intracellular targeting using green fluorescent protein, Western blot, and deletion analyses identified an N-terminal signal conveying mtKAS into mitochondria. Thus, mtKAS with its broad chain length specificity accomplishes all condensation steps in mitochondrial fatty acid synthesis, whereas in plastids three KAS enzymes are required.     

Molecular aspects of beta-ketoacyl synthase (KAS) catalysis

Crystal structure data for Escherichia coli beta-ketoacyl synthase (KAS) I with C(10) and C(12) fatty acid substrates bound in conjunction with results from mutagenizing residues in the active site leads to a model for catalysis. Differences from and similarities to the other Claisen enzymes carrying out decarboxylations reveal two catalytic mechanisms, one for KAS I and KAS II, the other for KAS III and chalcone synthase. A comparison of the structures of KAS I and KAS II does not reveal the basis of chain-length specificity. The structures of the Arabidopsis thaliana KAS family are compared.     

Beta-ketoacyl-acyl carrier protein synthase III (FabH) is essential for bacterial fatty acid synthesis

beta-Ketoacyl-acyl carrier protein (ACP) synthase III (KAS III, also called acetoacetyl-ACP synthase) encoded by the fabH gene is thought to catalyze the first elongation reaction (Claisen condensation) of type II fatty acid synthesis in bacteria and plant plastids. However, direct in vivo evidence that KAS III catalyzes an essential reaction is lacking, because no mutant organism deficient in this activity has been isolated. We report the first bacterial strain lacking KAS III, a fabH mutant constructed in the Gram-positive bacterium Lactococcus lactis subspecies lactis IL1403. The mutant strain carries an in-frame deletion of the KAS III active site region and was isolated by gene replacement using a medium supplemented with a source of saturated and unsaturated long-chain fatty acids. The mutant strain is devoid of KAS III activity and fails to grow in the absence of supplementation with exogenous long-chain fatty acids demonstrating that KAS III plays an essential role in cellular metabolism. However, the L. lactis fabH deletion mutant requires only long-chain unsaturated fatty acids for growth, a source of long-chain saturated fatty acids is not required. Because both saturated and unsaturated fatty acids are required for growth when fatty acid synthesis is blocked by biotin starvation (which prevents the synthesis of malonyl-CoA), another pathway for saturated fatty acid synthesis must remain in the fabH deletion strain. Indeed, incorporation of [1-14C]acetate into fatty acids in vivo showed that the fabH mutant retained about 10% of the fatty acid synthetic ability of the wild-type strain and that this residual synthetic capacity was preferentially diverted to the saturated branch of the pathway. Moreover, mass spectrometry showed that the fabH mutant retained low levels of palmitic acid upon fatty acid starvation. Derivatives of the fabH deletion mutant strain were isolated that were octanoic acid auxotrophs consistent with biochemical studies indicating that the major role of FabH is production of short-chain fatty acid primers. We also confirmed the essentiality of FabH in Escherichia coli by use of a plasmid-based gene insertion/deletion system. Together these results provide the first genetic evidence demonstrating that FabH conducts the major condensation reaction in the initiation of type II fatty acid biosynthesis in both Gram-positive and Gram-negative bacteria.     

The initiating steps of a type II fatty acid synthase in Plasmodium falciparum are catalyzed by pfACP,pfMCAT, and pfKASIII

Malaria, a disease caused by protozoan parasites of the genus Plasmodium, is one of the most dangerous infectious diseases, claiming millions of lives and infecting hundreds of millions of people annually. The pressing need for new antimalarials has been answered by the discovery of new drug targets from the malaria genome project. One of the early findings was the discovery of two genes encoding Type II fatty acid biosynthesis proteins: ACP (acyl carrier protein) and KASIII (beta-ketoacyl-ACP synthase III). The initiating steps of a Type II system require a third protein: malonyl-coenzyme A:ACP transacylase (MCAT). Here we report the identification of a single gene from P. falciparum encoding pfMCAT and the functional characterization of this enzyme. Pure recombinant pfMCAT catalyzes malonyl transfer from malonyl-coenzyme A (malonyl-CoA) to pfACP. In contrast, pfACP(trans), a construct of pfACP containing an amino-terminal apicoplast transit peptide, was not a substrate for pfMCAT. The product of the pfMCAT reaction, malonyl-pfACP, is a substrate for pfKASIII, which catalyzes the decarboxylative condensation of malonyl-pfACP and various acyl-CoAs. Consistent with a role in de novo fatty acid biosynthesis, pfKASIII exhibited typical KAS (beta-ketoacyl ACP synthase) activity using acetyl-CoA as substrate (k(cat) 230 min(-1), K(M) 17.9 +/- 3.4 microM). The pfKASIII can also catalyze the condensation of malonyl-pfACP and butyryl-CoA (k(cat) 200 min(-1), K(M) 35.7 +/- 4.4 microM) with similar efficiency, whereas isobutyryl-CoA is a poor substrate and displayed 13-fold less activity than that observed for acetyl-CoA. The pfKASIII has little preference for malonyl-pfACP (k(cat)/K(M) 64.9 min(-1)microM(-1)) over E. coli malonyl-ACP (k(cat)/K(M) 44.8 min(-1)microM(-1)). The pfKASIII also catalyzes the acyl-CoA:ACP transacylase (ACAT) reaction typically exhibited by KASIII enzymes, but does so almost 700-fold slower than the KAS reaction. Thiolactomycin did not inhbit pfKASIII (IC(50) > 330 microM), but three structurally similar substituted 1,2-dithiole-3-one compounds did inhibit pfKASIII with IC(50) values between 0.53 microM and 10.4 microM. These compounds also inhibited the growth of P. falciparum in culture.     

X-ray crystal structure of Mycobacterium tuberculosis beta-ketoacyl acyl carrier protein synthase II (mtKasB)

Mycolic acids are long chain alpha-alkyl branched, beta-hydroxy fatty acids that represent a characteristic component of the Mycobacterium tuberculosis cell wall. Through their covalent attachment to peptidoglycan via an arabinogalactan polysaccharide, they provide the basis for an essential outer envelope membrane. Mycobacteria possess two fatty acid synthases (FAS); FAS-I carries out de novo synthesis of fatty acids while FAS-II is considered to elongate medium chain length fatty acyl primers to provide long chain (C(56)) precursors of mycolic acids. Here we report the crystal structure of Mycobacterium tuberculosis beta-ketoacyl acyl carrier protein synthase (ACP) II mtKasB, a mycobacterial elongation condensing enzyme involved in FAS-II. This enzyme, along with the M. tuberculosis beta-ketoacyl ACP synthase I mtKasA, catalyzes the Claisen-type condensation reaction responsible for fatty acyl elongation in FAS-II and are potential targets for development of novel anti-tubercular drugs. The crystal structure refined to 2.4 A resolution revealed that, like other KAS-II enzymes, mtKasB adopts a thiolase fold but contains unique structural features in the capping region that may be crucial to its preference for longer fatty acyl chains than its counterparts from other bacteria. Modeling of mtKasA using the mtKasB structure as a template predicts the overall structures to be almost identical, but a larger entrance to the active site tunnel is envisaged that might contribute to the greater sensitivity of mtKasA to the inhibitor thiolactomycin (TLM). Modeling of TLM binding in mtKasB shows that the drug fits the active site poorly and results of enzyme inhibition assays using TLM analogues are wholly consistent with our structural observations. Consequently, the structure described here further highlights the potential of TLM as an anti-tubercular lead compound and will aid further exploration of the TLM scaffold towards the design of novel compounds, which inhibit mycobacterial KAS enzymes more effectively.     

Structure of the human beta-ketoacyl [ACP] synthase from the mitochondrial type II fatty acid synthase

Two distinct ways of organizing fatty acid biosynthesis exist: the multifunctional type I fatty acid synthase (FAS) of mammals, fungi, and lower eukaryotes with activities residing on one or two polypeptides; and the dissociated type II FAS of prokaryotes, plastids, and mitochondria with individual activities encoded by discrete genes. The beta-ketoacyl [ACP] synthase (KAS) moiety of the mitochondrial FAS (mtKAS) is targeted by the antibiotic cerulenin and possibly by the other antibiotics inhibiting prokaryotic KASes: thiolactomycin, platensimycin, and the alpha-methylene butyrolactone, C75. The high degree of structural similarity between mitochondrial and prokaryotic KASes complicates development of novel antibiotics targeting prokaryotic KAS without affecting KAS domains of cytoplasmic FAS. KASes catalyze the C(2) fatty acid elongation reaction using either a Cys-His-His or Cys-His-Asn catalytic triad. Three KASes with different substrate specificities participate in synthesis of the C(16) and C(18) products of prokaryotic FAS. By comparison, mtKAS carries out all elongation reactions in the mitochondria. We present the X-ray crystal structures of the Cys-His-His-containing human mtKAS and its hexanoyl complex plus the hexanoyl complex of the plant mtKAS from Arabidopsis thaliana. The structures explain (1) the bimodal (C(6) and C(10)-C(12)) substrate preferences leading to the C(8) lipoic acid precursor and long chains for the membranes, respectively, and (2) the low cerulenin sensitivity of the human enzyme; and (3) reveal two different potential acyl-binding-pocket extensions. Rearrangements taking place in the active site, including subtle changes in the water network, indicate a change in cooperativity of the active-site histidines upon primer binding.     

Beta-ketoacyl-acyl carrier protein synthase IV: a key enzyme for regulation of medium-chain fatty acid synthesis in Cuphea lanceolata seeds

With the aim of elucidating the mechanisms involved in the biosynthesis of medium-chain fatty acids in Cuphea lanceolata Ait., a crop accumulating up to 90% decanoic acid in seed triacylglycerols, cDNA clones of a beta-ketoacyl-acyl carrier protein (ACP) synthase IV (clKAS IV, EC 2.3.1.41) were isolated from C. lanceolata seed embryos. The amino acid sequence deduced from clKAS IV cDNA showed 80% identity to other plant KAS II-type enzymes, 55% identity towards plant KAS I and over 90% towards other Cuphea KAS IV-type sequences. Recombinant clKAS IV was functionally overexpressed in Escherichia coli, and substrate specificity of purified enzyme showed strong preference for elongation of short-chain and medium-chain acyl-ACPs (C4- to C10-ACP) with nearly equal activity. Further elongation steps were catalysed with distinctly less activity. Moreover, short- and medium-chain acyl-ACPs exerted a chain-length-specific and concentration-dependent substrate inhibition of clKAS IV. Based on these findings a regulatory mechanism for medium-chain fatty acid synthesis in C. lanceolata is presented.     

Structures of beta-ketoacyl-acyl carrier protein synthase I complexed with fatty acids elucidate its catalytic machinery

BACKGROUND: beta-ketoacyl-acyl carrier protein synthase (KAS) I is vital for the construction of the unsaturated fatty acid carbon skeletons characterizing E. coli membrane lipids. The new carbon-carbon bonds are created by KAS I in a Claisen condensation performed in a three-step enzymatic reaction. KAS I belongs to the thiolase fold enzymes, of which structures are known for five other enzymes. RESULTS: Structures of the catalytic Cys-Ser KAS I mutant with covalently bound C10 and C12 acyl substrates have been determined to 2.40 and 1.85 A resolution, respectively. The KAS I dimer is not changed by the formation of the complexes but reveals an asymmetric binding of the two substrates bound to the dimer. A detailed model is proposed for the catalysis of KAS I. Of the two histidines required for decarboxylation, one donates a hydrogen bond to the malonyl thioester oxo group, and the other abstracts a proton from the leaving group. CONCLUSIONS: The same mechanism is proposed for KAS II, which also has a Cys-His-His active site triad. Comparison to the active site architectures of other thiolase fold enzymes carrying out a decarboxylation step suggests that chalcone synthase and KAS III with Cys-His-Asn triads use another mechanism in which both the histidine and the asparagine interact with the thioester oxo group. The acyl binding pockets of KAS I and KAS II are so similar that they alone cannot provide the basis for their differences in substrate specificity.     

beta-Ketoacyl-[acyl carrier protein] synthase I of Escherichia coli: aspects of the condensation mechanism revealed by analyses of mutations in the active site pocket

beta-Ketoacyl-[acyl carrier protein (ACP)] synthase forms new carbon-carbon bonds in three steps: transfer of an acyl primer from ACP to the enzyme, decarboxylation of the elongating substrate and its condensation with the acyl primer substrate. Six residues of Escherichia coli beta-ketoacyl-ACP synthase I (KAS I) implicated in these reactions were subjected to site-directed mutagenesis. Analyses of the abilities of C163A, C163S, H298A, D306A, E309A, K328A, and H333A to carry out the three reactions lead to the following conclusions. The active site Cys-163 is not required for decarboxylation, whereas His-298 and His-333 are indispensable. Neither of the histidines is essential for increasing the nucleophilicity of Cys-163 to enable transfer of the acyl primer substrate. Maintenance of the structural integrity of the active site by Asp-306 and Glu-309 is required for decarboxylation but not for transfer. One function of Lys-328 occurs very early in catalysis, potentially before transfer. These results in conjunction with structural analyses of substrate complexes have led to a model for KAS I catalysis [Olsen, J. G., Kadziola, A., von Wettstein-Knowles, P., Siggaard-Andersen, M., and Larsen, S. (2001) Structure 9, 233-243]. Another facet of catalysis revealed by the mutant analyses is that the acyl primer transfer activity of beta-ketoacyl-ACP synthase I is inhibited by free ACP at physiological concentrations. Differences in the inhibitory response by individual mutant proteins indicate that interaction of free ACP with Cys-163, Asp-306, Glu-309, Lys-328, and His-333 might form a sensitive regulatory mechanism for the transfer of acyl primers.     

Fatty Acid Biosynthesis in Pseudomonas aeruginosa Is Initiated by the FabY Class of β-Ketoacyl Acyl Carrier Protein Synthases

The prototypical type II fatty acid synthesis (FAS) pathway in bacteria utilizes two distinct classes of β-ketoacyl synthase (KAS) domains to assemble long-chain fatty acids, the KASIII domain for initiation and the KASI/II domain for elongation. The central role of FAS in bacterial viability and virulence has stimulated significant effort toward developing KAS inhibitors, particularly against the KASIII domain of the β-acetoacetyl-acyl carrier protein (ACP) synthase FabH. Herein, we show that the opportunistic pathogen Pseudomonas aeruginosa does not utilize a FabH ortholog but rather a new class of divergent KAS I/II enzymes to initiate the FAS pathway. When a P. aeruginosa cosmid library was used to rescue growth in a fabH downregulated strain of Escherichia coli, a single unannotated open reading frame, PA5174, complemented fabH depletion. While deletion of all four KASIII domain-encoding genes in the same P. aeruginosa strain resulted in a wild-type growth phenotype, deletion of PA5174 alone specifically attenuated growth due to a defect in de novo FAS. Siderophore secretion and quorum-sensing signaling, particularly in the rhl and Pseudomonas quinolone signal (PQS) systems, was significantly muted in the absence of PA5174. The defect could be repaired by intergeneric complementation with E. coli fabH. Characterization of recombinant PA5174 confirmed a preference for short-chain acyl coenzyme A (acyl-CoA) substrates, supporting the identification of PA5174 as the predominant enzyme catalyzing the condensation of acetyl coenzyme A with malonyl-ACP in P. aeruginosa. The identification of the functional role for PA5174 in FAS defines the new FabY class of β-ketoacyl synthase KASI/II domain condensation enzymes.     

Biochemical characterization of 3-ketoacyl-acyl carrier protein synthase II from leek epidermis

In order to define its possible involvement in production of stearic acid for wax biosynthesis, the presence of 3-ketoacyl acyl synthase II (KAS II) activity was investigated in different tissues of leek (Allium porrum L.) leaves. KAS II activity was identified in sheath and lamina epidermis, as well as in underlying parenchyma. In all three tissues, activity was inhibited by 50% on addition of 100 microM cerulenin, and showed an absolute requirement for acyl-ACP substrates. More interestingly, the different tissues did not display similar KAS II substrate specificities. Parenchyma and lamina epidermis tissues presented typical KAS II activities, since C(18:0)-ACP was the exclusive product. In contrast, in sheath epidermis, KAS II activity resulted in the synthesis of acyl-chains up to 22 carbons in length, suggesting the existence in this tissue of an unusual KAS II. This activity was sufficient to elongate all of the palmitoyl-ACP produced by the fatty acid synthase, suggesting that C(18:0) is the substrate of the microsomal elongases involved in wax biosynthesis.     

Condensing enzymes from Cuphea wrightii associated with medium chain fatty acid biosynthesis

Seed oils of most Cuphea species contain > 90% medium chain (C8–C14) fatty acids. Thioesterases with specificity for these substrates are important determinants of the medium chain phenotype. The role of condensing enzymes, however, has not been investigated. cDNA clones encoding β-ketoacyl-acyl carrier protein (ACP) synthase (KAS) were isolated from C. wrightii, a C10/C12-producing species. Deduced amino acid sequences of four unique clones were ～ 60% identical to plant KAS I sequences and ～ 75% identical to a distinct class of KAS sequences recently identified in castor and barley. A 46 kDa protein that was observed only in developing and mature seed was detected using antiserum directed against recombinant Cuphea KAS protein. The 46 kDa protein was abundant in developing seeds of six medium chain-producing Cuphea species but barely detected in one long chain-producing species. A 48 kDa protein identified immunologically as KAS I was expressed in both medium and long chain-producing Cuphea species and was detected in all tissues tested. In in vitro assays, extracts from C. wrightii and C. viscosissima developing embryos were unable to extend fatty acid chains beyond C10 following treatment with 10 μm cerulenin, a potent inhibitor of KAS I. However, a C. viscosissima mutant, cpr-1, whose seed oils are deficient in caprate relative to wild type, was impaired in extension of C8 to C10 in this assay and Western analysis revealed a specific deficiency in 46 kDa KAS in cpr-1 embryos. These results implicate cerulenin-resistant condensing activity in production of medium chain fatty acids in Cuphea.     

Knockout of the regulatory site of 3-ketoacyl-ACP synthase III enhances short- and medium-chain acyl-ACP synthesis

3-ketoacyl-acyl carrier protein synthase (KAS) III catalyses the first condensing step of the fatty acid synthase (FAS) type II reaction in plants and bacteria, using acetyl CoA and malonyl-acyl carrier protein (ACP) as substrates. Enzymatic characterization of recombinant KAS III from Cuphea wrightii embryo shows that this enzyme is strongly inhibited by medium-chain acyl-ACP end products of the FAS reaction, i.e. inhibition by lauroyl-ACP was uncompetitive towards acetyl CoA and non-competitive with regard to malonyl-ACP. This indicated a distinct attachment site for regulatory acyl-ACPs. Based on alignment of primary structures of various KAS IIIs and 3-ketoacyl CoA synthases, we suspected the motif G290NTSAAS296 to be responsible for binding of regulatory acyl-ACPs. Deletion of the tetrapeptide G290NTS293 led to a change of secondary structure and complete loss of KAS III condensing activity. Exchange of asparagine291 to aspartate, alanine294 to serine and alanine295 to proline, however, produced mutant enzymes with slightly reduced condensing activity, yet with insensitivity towards acyl-ACPs. To assess the potential of unregulated KAS III as tool in oil production, we designed in vitro experiments employing FAS preparations from medium-chain fatty acid-producing Cuphea lanceolata seeds and long-chain fatty acid-producing rape seeds, each supplemented with a fivefold excess of the N291D KAS III mutant. High amounts of short-chain acyl-ACPs in the case of C. lanceolata, and of medium-chain acyl-ACPs in the case of rape seed preparations, were obtained. This approach targets regulation and offers new possibilities to derive transgenic or non-transgenic plants for production of seed oils with new qualities.     

Purification and biochemical characterization of lysosomal acid phosphatases (EC 3.1.3.2) from blood stream forms, Trypanosoma brucei brucei

Three Acid phosphatases (ACP) were isolated and characterized from the lysosomes of blood stream forms of Trypanosoma brucei by a combination of isopynic and differential centrifugation through Ficoll, organic solvent precipitation, ion exchange on DEAE cellulose 52 and size exclusion chromatography on Sephadex G-75 columns. The purified ACP emerged as three distinct peaks (ACP I, ACP II and ACP III) with high specific activities and they moved homogenously on 12% SDS-PAGE each as a single band with relative molecular weight of 36 kDa, 25 kDa and 45 kDa respectively. The purified enzymes were active at an optimum pH and temperature of 5.5 and 40 °C respectively. The enzyme activities appeared to be ACP because their activities were enhanced at low pH values and inhibited by the acid phosphatase inhibitor, sodium fluoride. ACP I and ACP II were sensitive to l-tartrate while ACP III was insensitive to l tartrate. The kinetic analysis of the purified enzyme (ACP I, ACP II and ACP III) determined using para-nitrophenylphosphate as substrate gave K_M values of 0.2 mM, 0.15 mM and 0.5 mM. Monofunctional group sulfhydryl group inhibitors; HgCl₂, and AgCl₂ strongly inhibited the activity of ACP III and millimolar concentrations of dithiothreitol and iodoacetamide activated and inhibited the activity of the ACP III respectively, suggesting the involvement of thiol groups at the active site of the enzyme. Thus, differentiating it from ACP I and ACP II. The implication of these findings in relation to the pathology of trypanosomosis is discussed.     

Identification,characterization, and inhibition of Plasmodium falciparum beta-hydroxyacyl-acyl carrier protein dehydratase (FabZ)

The emergence of drug-resistant forms of Plasmodium falciparum emphasizes the need to develop new antimalarials. In this context, the fatty acid biosynthesis (FAS) pathway of the malarial parasite has recently received a lot of attention. Due to differences in the fatty acid biosynthesis systems of Plasmodium and man, this pathway is a good target for the development of new and selective therapeutic drugs directed against malaria. In continuation of these efforts we report cloning and overexpression of P. falciparum beta-hydroxyacyl-acyl carrier protein (ACP) dehydratase (PffabZ) gene that codes for a 17-kDa protein. The enzyme catalyzes the dehydration of beta-hydroxyacyl-ACP to trans-2-acyl-ACP, the third step in the elongation phase of the FAS cycle. It has a Km of 199 microM and kcat/Km of 80.4 m-1 s-1 for the substrate analog beta-hydroxybutyryl-CoA but utilizes crotonoyl-CoA, the product of the reaction, more efficiently (Km = 86 microM, kcat/Km = 220 m-1 s-1). More importantly, we also identify inhibitors (NAS-91 and NAS-21) for the enzyme. Both the inhibitors prevented the binding of crotonoyl-CoA to PfFabZ in a competitive fashion. Indeed these inhibitors compromised the growth of P. falciparum in cultures and inhibited the parasite fatty acid synthesis pathway both in cell-free extracts as well as in situ. We modeled the structure of PfFabZ using Escherichia coli beta-hydroxydecanoyl thioester dehydratase (EcFabA) as a template. We also modeled the inhibitor complexes of PfFabZ to elucidate the mode of binding of these compounds to FabZ. The discovery of the inhibitors of FabZ, reported for the first time against any member of this family of enzymes, essential to the type II FAS pathway opens up new avenues for treating a number of infectious diseases including malaria.     

Identification of a plastid acyl-acyl carrier protein synthetase in Arabidopsis and its role in the activation and elongation of exogenous fatty acids

Plant cells are known to elongate exogenously provided fatty acid (FA), but the subcellular sites and mechanisms for this process are not currently understood. When Arabidopsis leaves were incubated with 14C-FAs with or=20 carbons) but not synthesis of 14C-unsaturated 18-carbon or 16-carbon FAs. Isolated pea chloroplasts were also able to elongate 14C-FAs (相似文献   

Enzymes involved in fatty acid and polyketide biosynthesis in Streptomyces glaucescens: role of FabH and FabD and their acyl carrier protein specificity

Malonyl acyl carrier protein (ACP) is used as an extender unit in each of the elongation steps catalyzed by the type II dissociated fatty acid synthase (FAS) and polyketide synthase (PKS) of Streptomyces glaucescens. Initiation of straight-chain fatty acid biosynthesis by the type II FAS involves a direct condensation of acetyl-CoA with this malonyl-ACP to generate a 3-ketobutyryl-ACP product and is catalyzed by FabH. In vitro experiments with a reconstituted type II PKS system in the absence of FabH have previously shown that the acetyl-ACP (generated by decarboxylation of malonyl-ACP), not acetyl-CoA, is used to initiate tetracenomycin C (TCM C) biosynthesis. We have shown that sgFabH activity is present in S. glaucescens fermentations during TCM C production, suggesting that it could contribute to initiation of TCM C biosynthesis in vivo. Isotope incorporation studies with [CD3]acetate and [13CD3]acetate demonstrated significant intact retention of three deuteriums into the starter unit of palmitate and complete washout of deuterium label into the starter unit of TCM C. These observations provide evidence that acetyl-CoA is not used directly as a starter unit for TCM C biosynthesis in vivo and argue against an involvement of FabH in this process. Consistent with this conclusion, assays of the purified recombinant sgFabH with acetyl-CoA demonstrated activity using malonyl-ACP generated from either FabC (the S. glaucescens FAS ACP) (k(cat) 42.2 min(-1), K(m) 4.5 +/- 0.3 microM) or AcpP (the E. coli FAS ACP) (k(cat) 7.5 min(-1), K(m) 6.3 +/- 0.3 microM) but not TcmM (the S. glaucescens PKS ACP). In contrast, the sgFabD which catalyzes conversion of malonyl-CoA to malonyl-ACP for fatty acid biosynthesis was shown to be active with TcmM (k(cat) 150 min(-1), K(m) 12.2 +/- 1.2 microM), AcpP (k(cat) 141 min(-1), K(m) 13.2 +/- 1.6 microM), and FabC (k(cat) 560 min(-1), K(m) 12.7 +/- 2.6 microM). This enzyme was shown to be present during TCM C production and could play a role in generating malonyl-ACP for both processes. Previous demonstrations that the purified PKS ACPs catalyze self-malonylation and that a FabD activity is not required for polyketide biosynthesis are shown to be an artifact of the expression and purification protocols. The relaxed ACP specificity of FabD and the lack of a clear alternative are consistent with a role of FabD in providing malonyl-ACP precursors for PKS as well as FAS processes. In contrast, the ACP specificity of FabH, isotope labeling studies, and a demonstrated alternative mechanism for initiation of the PKS process provide unequivocal evidence that FabH is involved only in the FAS process.     

A template search reveals mechanistic similarities and differences in beta-ketoacyl synthases (KAS) and related enzymes

A detailed comparison of the active sites in beta-ketoacyl synthases (KAS) and related enzymes has been made. Using three-dimensional templates of the three catalytic residues to scan the protein structural database reveals differences in both the geometry and the catalytic role of equivalent residues in different members of the family. The template based on the catalytic cysteine and two histidines in the KAS I and II is totally specific for this family, with no false hits. However, the role of the histidines in catalysis is different between KAS I/II and thiolase on the one hand and KAS III/chalcone synthase on the other. In contrast, a template comprising only cysteine and one histidine is not specific with many hits including members of the KAS family, metal binding sites, other active sites in nonhomologous proteins, and some "random" nonactive sites.