Structure of beta-ketoacyl-[acyl carrier protein] reductase from Escherichia coli: negative cooperativity and its structural basis.

The structure of beta-ketoacyl-[acyl carrier protein] reductase (FabG) from Escherichia coli was determined via the multiwavelength anomalous diffraction technique using a selenomethionine-labeled crystal containing 88 selenium sites in the asymmetric unit. The comparison of the E. coli FabG structure with the homologous Brassica napus FabG.NADP(+) binary complex reveals that cofactor binding causes a substantial conformational change in the protein. This conformational change puts all three active-site residues (Ser 138, Tyr 151, and Lys 155) into their active configurations and provides a structural mechanism for allosteric communication between the active sites in the homotetramer. FabG exhibits negative cooperative binding of NADPH, and this effect is enhanced by the presence of acyl carrier protein (ACP). NADPH binding also increases the affinity and decreases the maximum binding of ACP to FabG. Thus, unlike other members of the short-chain dehydrogenase/reductase superfamily, FabG undergoes a substantial conformational change upon cofactor binding that organizes the active-site triad and alters the affinity of the other substrate-binding sites in the tetrameric enzyme.     

Deciphering the key residues in Plasmodium falciparum beta-ketoacyl acyl carrier protein reductase responsible for interactions with Plasmodium falciparum acyl carrier protein

The type II fatty acid synthase (FAS) pathway of Plasmodium falciparum is a validated unique target for developing novel antimalarials, due to its intrinsic differences from the typeI pathway operating in humans. beta-Ketoacyl acyl carrier protein (ACP) reductase (FabG) performs the NADPH-dependent reduction of beta-ketoacyl-ACP to beta-hydroxyacyl-ACP, the first reductive step in the elongation cycle of fatty acid biosynthesis. In this article, we report intensive studies on the direct interactions of Plasmodium FabG and Plasmodium ACP in solution, in the presence and absence of its cofactor, NADPH, by monitoring the change in intrinsic fluorescence of P.falciparum FabG (PfFabG) and by surface plasmon resonance. To address the issue of the importance of the residues involved in strong, specific and stoichiometric binding of PfFabG to P.falciparum ACP (PfACP), we mutated Arg187, Arg190 and Arg230 of PfFabG. The activities of the mutants were assessed using both an ACP-dependent and an ACP-independent assay. The affinities of all the PfFabG mutants for acetoacetyl-ACP (the physiological substrate) were reduced to different extents as compared to wild-type PfFabG, but were equally active in biochemical assays with the substrate analog acetoacetyl-CoA. Kinetic analysis and studies of direct binding between PfFabG and PfACP confirmed the identification of Arg187 and Arg230 as critical residues for the PfFabG-PfACP interactions. Our studies thus reveal the significance of the positively charged/hydrophobic patch located adjacent to the active site cavities of PfFabG for interactions with PfACP.     

The X-ray structure of Brassica napus beta-keto acyl carrier protein reductase and its implications for substrate binding and catalysis

BACKGROUND: beta-Keto acyl carrier protein reductase (BKR) catalyzes the pyridine-nucleotide-dependent reduction of a 3-oxoacyl form of acyl carrier protein (ACP), the first reductive step in de novo fatty acid biosynthesis and a reaction often performed in polyketide biosynthesis. The Brassica napus BKR enzyme is NADPH-dependent and forms part of a dissociable type II fatty acid synthetase (FAS). Significant sequence similarity is observed with enoyl acyl carrier protein reductase (ENR), the other reductase of FAS, and the short-chain alcohol dehydrogenase (SDR) family. RESULTS: The first crystal structure of BKR has been determined at 2.3 A resolution in a binary complex with an NADP(+) cofactor. The structure reveals a homotetramer in which each subunit has a classical dinucleotide-binding fold. A triad of Ser154, Tyr167 and Lys171 residues is found at the active site, characteristic of the SDR family. Overall BKR has a very similar structure to ENR with good superimposition of catalytically important groups. Modelling of the substrate into the active site of BKR indicates the need for conformational changes in the enzyme. CONCLUSIONS: A catalytic mechanism can be proposed involving the conserved triad. Helix alpha6 must shift its position to permit substrate binding to BKR and might act as a flexible lid on the active site. The similarities in fold, mechanism and substrate binding between BKR, which catalyzes a carbon-oxygen double-bond reduction, and ENR, the carbon-carbon double-bond oxidoreductase in FAS, suggest a close evolutionary link during the development of the fatty acid biosynthetic pathway.     

Lipid biosynthesis in developing and germinating soybean cotyledons: The formation of oleate by a soluble stearyl acyl carrier protein desaturase

Extracts of developing soybean cotyledons contain a highly specific stearyl acyl carrier protein (ACP) desaturase which in the presence of NADPH, O₂, ferredoxin and ferredoxin: NADP⁺ reductase, rapidly converts stearyl ACP to oleyl ACP. The enzyme system has a high affinity for O₂, near-maximal activity being obtained at only 10 μm O₂. The pH optimum for the desaturase is 6.0. Stearic acid and stearyl-CoA, alone or in the presence of acyl carrier protein, are totally inactive. Although the enzyme is found in extracts prepared from developing soybean seeds (15–50 days after flowering), activity was not detected in extracts of germinated seeds.     

2-Acylglycerolphosphoethanolamine acyltransferase/acyl-acyl carrier protein synthetase is a membrane-associated acyl carrier protein binding protein

Two enzymatic activities, 2-acylglycerolphosphoethanolamine (2-acyl-GPE) acyltransferase and acyl-acyl carrier protein (acyl-ACP) synthetase, were solubilized and purified from Escherichia coli membranes. Electrophoretic analysis of the final product of the purification procedure revealed a single protein species with an apparent molecular mass of 27 kilodaltons. The ratio of acyltransferase to synthetase activities remained the same throughout the purification scheme indicating that both activities were catalyzed by the same enzyme. 2-Acyl-GPE acyltransferase exhibited an apparent ACP Km of 64 nM under standard assay conditions that increased to 10 microM when the assay was conducted in the presence of 0.4 M LiCl. Acyl-ACP synthetase activity was not detected in the absence of 0.4 M LiCl, and the apparent ACP Km for this reaction was 16 microM. Direct evidence that ACP was a subunit of the acyltransferase/synthetase was obtained by the adsorption of both catalytic activities to an ACP-Sepharose affinity column and by the binding of [3H]ACP to the purified enzyme preparation. The apparent Km for acyl-ACP was 13 microM, and the rate of acyl transfer from this acyl donor was enhanced by the addition of 0.4 M LiCl indicating that the exchange of enzyme-bound ACP for acyl-ACP was a determinant factor in the rate of phosphatidylethanolamine formation from acyl-ACP. These data indicate that the 2-acyl-GPE acyltransferase and acyl-ACP synthetase reactions are catalyzed by the same membrane protein that possesses a high affinity binding site for soluble ACP.     

细菌3-酮脂酰ACP还原酶研究进展

3-酮脂酰ACP还原酶(FabG)在细菌中广泛存在并且十分保守,已经发现的所有FabG及其同系物都具有类似的催化活性中心序列,隶属于短链醇脱氢酶/还原酶(SDRs)超家族成员。它是Ⅱ型脂肪酸合成反应中的关键酶,将3-酮脂酰ACP还原为3-羟脂酰ACP多以NADPH作为辅酶。从搜集的文献来看,国内外针对不同细菌中3-酮脂酰ACP还原酶同系物的研究报道体现了其多样性的特点。但是,近年来,该方面的专题综述十分少见。本文主要对3-酮脂酰ACP还原酶的结构特征、在脂肪酸合成和其他方面的生物学功能,以及以该酶为作用靶点的抑菌剂等方面进行概述,以期为将来3-酮脂酰ACP还原酶的深入研究提供理论参考。     

Inhibition of the Mycobacterium tuberculosis enoyl acyl carrier protein reductase InhA by arylamides

InhA, the enoyl acyl carrier protein reductase (ENR) from Mycobacterium tuberculosis, is one of the key enzymes involved in the type II fatty acid biosynthesis pathway of M. tuberculosis. We report here the discovery, through high-throughput screening, of a series of arylamides as a novel class of potent InhA inhibitors. These direct InhA inhibitors require no mycobacterial enzymatic activation and thus circumvent the resistance mechanism to antitubercular prodrugs such as INH and ETA that is most commonly observed in drug-resistant clinical isolates. The crystal structure of InhA complexed with one representative inhibitor reveals the binding mode of the inhibitor within the InhA active site. Further optimization through a microtiter synthesis strategy followed by in situ activity screening led to the discovery of a potent InhA inhibitor with in vitro IC(50)=90 nM, representing a 34-fold potency improvement over the lead compound.     

Fat metabolism in higher plants. Properties of the palmityl acyl carrier protein: stearyl acyl carrier protein elongation system in maturing safflower seed extracts

Palmityl acyl carrier protein is elongated specifically to stearyl acyl carrier protein by a system which required palmityl acyl carrier protein, malonyl CoA, and NADPH. Extracts from maturing safflower seeds, avocado mesocarp, and stroma from spinach chloroplasts contain the elongation system. The system differs from the de novo fatty acid synthetase system in that (1) it is inactivated at 37 °C whereas the de novo system remains fully active, (2) the pH optimum of the elongation system is 7.8–8.6 whereas the de novo system has a narrow pH optimum at 7.0, (3) NADPH is specifically required whereas the de novo system requires both NADPH and NADH, and (4) the elongation system is relatively insensitive to cerulenin whereas the de novo system is highly sensitive. Acetyl CoA does not serve as a C₂ donor. Stearyl acyl carrier protein, lauryl CoA, myristyl CoA, and palmityl CoA are inactive.     

Structural elucidation of the specificity of the antibacterial agent triclosan for malarial enoyl acyl carrier protein reductase.

The human malaria parasite Plasmodium falciparum synthesizes fatty acids using a type II pathway that is absent in humans. The final step in fatty acid elongation is catalyzed by enoyl acyl carrier protein reductase, a validated antimicrobial drug target. Here, we report the cloning and expression of the P. falciparum enoyl acyl carrier protein reductase gene, which encodes a 50-kDa protein (PfENR) predicted to target to the unique parasite apicoplast. Purified PfENR was crystallized, and its structure resolved as a binary complex with NADH, a ternary complex with triclosan and NAD(+), and as ternary complexes bound to the triclosan analogs 1 and 2 with NADH. Novel structural features were identified in the PfENR binding loop region that most closely resembled bacterial homologs; elsewhere the protein was similar to ENR from the plant Brassica napus (root mean square for Calphas, 0.30 A). Triclosan and its analogs 1 and 2 killed multidrug-resistant strains of intra-erythrocytic P. falciparum parasites at sub to low micromolar concentrations in vitro. These data define the structural basis of triclosan binding to PfENR and will facilitate structure-based optimization of PfENR inhibitors.     

Plant holo-(acyl carrier protein) synthase.

1. An improved method was developed for the assay of plant holo-(acyl carrier protein) synthase activity, using Escherichia coli acyl-(acyl carrier protein) synthetase as a coupling enzyme. 2. Holo-(acyl carrier protein) synthase was partially purified from spinach (Spinacia oleracea) leaves by a combination of (NH4)2SO4 fractionation and anion-exchange and gel-permeation chromatography. 3. The partially purified enzyme had a pH optimum of 8.2 and Km values of 2 microM, 72 microM and 3 mM for apo-(acyl carrier protein), CoA and Mg2+ respectively. Synthase activity was inhibited in vitro by the reaction product 3',5'-ADP. 4. Results from the fractionation of spinach leaf and developing castor-oil-seed (Ricinus communis) endosperm cells were consistent with a cytosolic localization of holo-(acyl carrier protein) synthase activity in plant cells.     

Turnover of the 4'-phosphopantetheine prosthetic group of acyl carrier protein

Acyl carrier protein is an essential cofactor in fatty acid biosynthesis, and in contrast to the stability of the protein moiety during growth, its 4'-phosphopantetheine prosthetic group is metabolically active. The biosynthetic incorporation of deuterium into nonexchangeable positions of acyl carrier protein was found to enhance the sensitivity of the protein to pH-induced hydrodynamic expansion. This constitutional isotope effect was exploited to separate deuterated from normal acyl carrier protein by conformationally sensitive gel electrophoresis, thus providing the analytical framework for separating pre-existing (deuterated) from newly synthesized acyl carrier protein in pulse-chase experiments. The rate of acyl carrier protein prosthetic group turnover was found to depend on the intracellular concentration of coenzyme A. At low coenzyme A levels, prosthetic group turnover was four times faster than the rate of new acyl carrier protein biosynthesis but at the higher coenzyme A concentrations characteristic of logarithmic growth, turnover was an order of magnitude slower, amounting to approximately 25% of the acyl carrier protein pool per generation. These observations suggest that the acyl carrier protein prosthetic group turnover cycle may be related to coenzyme A metabolism rather than to lipid biosynthesis.     

A continuous coupled enzyme assay for bacterial malonyl-CoA:acyl carrier protein transacylase (FabD)

Bacterial malonyl-CoA:acyl carrier protein transacylase catalyzes the transfer of a malonyl moiety from malonyl-CoA to the free thiol group of the phosphopantetheine arm of acyl carrier protein. Malonyl-ACP, the product of this enzymatic reaction, is the key building block for de novo fatty acid biosynthesis. Here, we describe a continuous enzyme assay based on the coupling of the malonyl-CoA:acyl carrier protein transacylase reaction to alpha-ketoglutarate dehydrogenase (KDH). KDH-dependent consumption of the coenzyme A generated by malonyl-CoA:acyl carrier protein transacylase is accompanied by a reduction of nicotinamide adenine dinucleotide, oxidized (NAD(+)) to nicotinamide adenine dinucleotide, reduced. The rate of NAD(+) reduction is continuously monitored as a change in fluorescence using a microtiter plate reader. We show that this coupled enzyme assay is amenable to routine chemical compound screening.     

The acyl dihydroxyacetone phosphate pathway enzymes for glycerolipid biosynthesis are present in the yeast Saccharomyces cerevisiae.

The presence of the acyl dihydroxyacetone phosphate (acyl DHAP) pathway in yeasts was investigated by examining three key enzyme activities of this pathway in Saccharomyces cerevisiae. In the total membrane fraction of S. cerevisiae, we confirmed the presence of both DHAP acyltransferase (DHAPAT; Km = 1.27 mM; Vmax = 5.9 nmol/min/mg of protein) and sn-glycerol 3-phosphate acyltransferase (GPAT; Km = 0.28 mM; Vmax = 12.6 nmol/min/mg of protein). The properties of these two acyltransferases are similar with respect to thermal stability and optimum temperature of activity but differ with respect to pH optimum (6.5 for GPAT and 7.4 for DHAPAT) and sensitivity toward the sulfhydryl blocking agent N-ethylmaleimide. Total membrane fraction of S. cerevisiae also exhibited acyl/alkyl DHAP reductase (EC 1.1.1.101) activity, which has not been reported previously. The reductase has a Vmax of 3.8 nmol/min/mg of protein for the reduction of hexadecyl DHAP (Km = 15 microM) by NADPH (Km = 20 microM). Both acyl DHAP and alkyl DHAP acted as substrates. NADPH was the specific cofactor. Divalent cations and N-ethylmaleimide inhibited the enzymatic reaction. Reductase activity in the total membrane fraction from aerobically grown yeast cells was twice that from anaerobically grown cells. Similarly, DHAPAT and GPAT activities were also greater in aerobically grown yeast cells. The presence of these enzymes, together with the absence of both ether glycerolipids and the ether lipid-synthesizing enzyme (alkyl DHAP synthase) in S. cerevisiae, indicates that non-ether glycerolipids are synthesized in this organism via the acyl DHAP pathway.     

Tryptophan fluorescence reveals induced folding of Vibrio harveyi acyl carrier protein upon interaction with partner enzymes

We have introduced tryptophan as a local fluorescent probe to monitor the conformation of Vibrio harveyi acyl carrier protein (ACP), a small flexible protein that is unfolded at neutral pH but must undergo reversible conformational change during the synthesis and delivery of bacterial fatty acids. Consistent with known 3D structures of ACP, steady-state fluorescence and quenching experiments indicated that Trp at positions 46, 50, and 72 are buried in the hydrophobic core upon Mg(2+)-induced ACP folding, whereas residues 25 and 45 remain in a hydrophilic environment on the protein surface. Attachment of fatty acids to the phosphopantetheine prosthetic group progressively stabilized the folded conformation of all Trp-substituted ACPs, but longer chains (14:0) were less effective than medium chains (8:0) in shielding Trp from acrylamide quenching in the L46W protein. Interaction with ACP-dependent enzymes LpxA and holo-ACP synthase also caused folding of L46W; fluorescence quenching indicated proximity of Trp-45 in helix II of ACP in LpxA binding. Our results suggest that divalent cations and fatty acylation produce differing environments in the ACP core and also reveal enzyme partner-induced folding of ACP, a key feature of "natively unfolded" proteins.     

Effect of substrate binding loop mutations on the structure, kinetics, and inhibition of enoyl acyl carrier protein reductase from Plasmodium falciparum

Enoyl acyl carrier protein reductase (ENR), which catalyzes the final and rate limiting step of fatty acid elongation, has been validated as a potential drug target. Triclosan is known to be an effective inhibitor for this enzyme. We mutated the substrate binding site residue Ala372 of the ENR of Plasmodium falciparum (PfENR) to Methionine and Valine which increased the affinity of the enzyme towards triclosan to almost double, close to that of Escherichia coli ENR (EcENR) which has a Methionine at the structurally similar position of Ala372 of PfENR. Kinetic studies of the mutants of PfENR and the crystal structure analysis of the A372M mutant revealed that a more hydrophobic environment enhances the affinity of the enzyme for the inhibitor. A triclosan derivative showed a threefold increase in the affinity towards the mutants compared to the wild type, due to additional interactions with the A372M mutant as revealed by the crystal structure. The enzyme has a conserved salt bridge which stabilizes the substrate binding loop and appears to be important for the active conformation of the enzyme. We generated a second set of mutants to check this hypothesis. These mutants showed loss of function, except in one case, where the crystal structure showed that the substrate binding loop is stabilized by a water bridge network.     

Evidence for interactions of acyl carrier protein with glycerol-3-phosphate acyltransferase, an inner membrane protein of Escherichia coli

We [(1989) FEBS Lett., in press] have previously shown that membrane vesicles from Escherichia coli contain protein-binding sites for the acyl carrier protein (ACP). We report now that membrane vesicles prepared from a strain amplified for glycerol-3-phosphate acyltransferase (GPAT) contain a higher number of ACP-binding sites than the membrane vesicles prepared from a wild type strain. In addition, we show that GPAT is retained specifically on an ACP-Sepharose affinity column and that [3H]ACP binds to the enzyme solubilized by detergent. We conclude that GPAT, an inner membrane protein which catalyses the transesterification of a fatty acyl group from acyl coenzyme A or acyl ACP to glycerol-3-phosphate, possesses a binding site for ACP.     

Characterization of recombinant thioesterase and acyl carrier protein domains of chicken fatty acid synthase expressed in Escherichia coli

Fatty acid synthase of animal tissue is a multifunctional enzyme comprised of two identical subunits, each containing seven partial activities and a site for the prosthetic group, 4'-phosphopantetheine (acyl carrier protein). We have recently isolated cDNA clones of chicken fatty acid synthase coding for the dehydratase, enoyl reductase, beta-ketoacyl reductase, acyl carrier protein, and thioesterase domains (Chirala, S.S., Kasturi, R., Pazirandeh, M., Stolow, D.T., Huang, W.Y., and Wakil, S.J. (1989) J. Biol. Chem. 264, 3750-3757). To gain insight into the structure and function of the various domains, the portion of the cDNA coding for the acyl carrier protein and thioesterase domains was expressed in Escherichia coli by using an expression vector that utilizes the phage lambda PL promoter. The recombinant protein was efficiently expressed and purified to near homogeneity using anion-exchange and hydroxyapatite chromatography. As expected from the coding capacity of the cDNA expressed, the protein has a molecular weight of 43,000 and reacts with antithioesterase antibodies. The recombinant thioesterase was found to be enzymatically active and has the same substrate specificity and kinetic properties as the native enzyme of the multifunctional synthase. Treatment of the recombinant protein with alpha-chymotrypsin results in the cleavage of the acyl carrier protein and thioesterase domain junction sequence at exactly the same site as with native fatty acid synthase. The amino acid composition of the purified recombinant protein revealed the presence of 0.6 mol of beta-alanine/mol of protein, indicating partial pantothenylation of the recombinant acyl carrier protein domain. These results indicate that the expressed protein has a conformation similar to the native enzyme and that its folding into functionally active domains is independent of the remaining domains of the multifunctional synthase subunit. These conclusions are consistent with the proposal that the multifunctional synthase gene has evolved from fusion of component genes.     

Synthesis and biological activity of diaryl ether inhibitors of malarial enoyl acyl carrier protein reductase. Part 2: 2'-substituted triclosan derivatives

2'-Substituted analogs of triclosan have been synthesized to target inhibition of the key malarial enzyme Plasmodium falciparum enoyl acyl carrier protein reductase (PfENR). Many of these compounds exhibit good potency (EC50<500 nM) against in vitro cultures of drug-resistant and drug-sensitive strains of the P. falciparum parasite and modest (IC50=1-20 microM) potency against purified PfENR enzyme. Compared to triclosan, this survey of 2'-substituted derivatives has afforded gains in excess of 20- and 30-fold versus the 3D7 and Dd2 strains of parasite, respectively.     

The X-ray crystal structure of beta-ketoacyl [acyl carrier protein] synthase I

The crystal structure of the fatty acid elongating enzyme beta-ketoacyl [acyl carrier protein] synthase I (KAS I) from Escherichia coli has been determined to 2.3 A resolution by molecular replacement using the recently solved crystal structure of KAS II as a search model. The crystal contains two independent dimers in the asymmetric unit. KAS I assumes the thiolase alpha(beta)alpha(beta)alpha fold. Electrostatic potential distribution reveals an acyl carrier protein docking site and a presumed substrate binding pocket was detected extending the active site. Both subunits contribute to each substrate binding site in the dimer.     

Molecular dynamics simulations of the Apo-, Holo-, and acyl-forms of Escherichia coli acyl carrier protein

Acyl carrier protein (ACP) is an essential co-factor protein in fatty acid biosynthesis that shuttles covalently bound fatty acyl intermediates in its hydrophobic pocket to various enzyme partners. To characterize acyl chain-ACP interactions and their influence on enzyme interactions, we performed 19 molecular dynamics (MD) simulations of Escherichia coli apo-, holo-, and acyl-ACPs. The simulations were started with the acyl chain in either a solvent-exposed or a buried conformation. All four short-chain (< or = C10) and one long-chain (C16) unbiased acyl-ACP MD simulation show the transition of the solvent-exposed acyl chain into the hydrophobic pocket of ACP, revealing its pathway of acyl chain binding. Although the acyl chain resides inside the pocket, Thr-39 and Glu-60 at the entrance stabilize the phosphopantetheine linker through hydrogen bonding. Comparisons of the different ACP forms indicate that the loop region between helices II and III and the prosthetic linker may aid in substrate recognition by enzymes of fatty acid synthase systems. The MD simulations consistently show that the hydrophobic binding pocket of ACP is best suited to accommodate an octanoyl group and is capable of adjusting in size to accommodate chain lengths as long as decanoic acid. The simulations also reveal a second, novel binding mode of the acyl chains inside the hydrophobic binding pocket directed toward helix I. This study provides a detailed dynamic picture of acyl-ACPs that is in excellent agreement with available experimental data and, thereby, provides a new understanding of enzyme-ACP interactions.