Biochemical and molecular analyses of the Streptococcus pneumoniae acyl carrier protein synthase, an enzyme essential for fatty acid biosynthesis

Acyl carrier protein synthase (AcpS) is an essential enzyme in the biosynthesis of fatty acids in all bacteria. AcpS catalyzes the transfer of 4'-phosphopantetheine from coenzyme A (CoA) to apo-ACP, thus converting apo-ACP to holo-ACP that serves as an acyl carrier for the biosynthesis of fatty acids and lipids. To further understand the physiological role of AcpS, we identified, cloned, and expressed the acpS and acpP genes of Streptococcus pneumoniae and purified both products to homogeneity. Both acpS and acpP form operons with the genes whose functions are required for other cellular metabolism. The acpS gene complements an Escherichia coli mutant defective in the production of AcpS and appears to be essential for the growth of S. pneumoniae. Gel filtration and cross-linking analyses establish that purified AcpS exists as a homotrimer. AcpS activity was significantly stimulated by apo-ACP at concentrations over 10 microm and slightly inhibited at concentrations of 5-10 microm. Double reciprocal analysis of initial velocities of AcpS at various concentrations of CoA or apo-ACP indicated a random or compulsory ordered bi bi type of reaction mechanism. Further analysis of the inhibition kinetics of the product (3',5'-ADP) suggested that it is competitive with respect to CoA but mixed (competitive and noncompetitive) with respect to apo-ACP. Finally, apo-ACP bound tightly to AcpS in the absence of CoA, but CoA failed to do so in the absence of apo-ACP. Together, these results suggest that AcpS may be allosterically regulated by apo-ACP and probably proceeds by an ordered reaction mechanism with the first formation of the AcpS-apo-ACP complex and the subsequent transfer of 4'-phosphopantetheine to the apo-ACP of the complex.     

Crystal structure of beta-ketoacyl-acyl carrier protein synthase III. A key condensing enzyme in bacterial fatty acid biosynthesis

Beta-ketoacyl-acyl carrier protein synthase III (FabH), the most divergent member of the family of condensing enzymes, is a key catalyst in bacterial fatty acid biosynthesis and a promising target for novel antibiotics. We report here the crystal structures of FabH determined in the presence and absence of acetyl-CoA. These structures display a fold that is common for condensing enzymes. The observed acetylation of Cys(112) proves its catalytic role and clearly defines the primer binding pocket. Modeling based on a bound CoA molecule suggests catalytic roles for His(244) and Asn(274). The structures provide the molecular basis for FabH substrate specificity and reaction mechanism and are important for structure-based design of novel antibiotics.     

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.     

Crystal structure of Mycobacterium tuberculosis diaminopimelate decarboxylase,an essential enzyme in bacterial lysine biosynthesis

The Mycobacterium tuberculosis lysA gene encodes the enzyme meso-diaminopimelate decarboxylase (DAPDC), a pyridoxal-5'-phosphate (PLP)-dependent enzyme. The enzyme catalyzes the final step in the lysine biosynthetic pathway converting meso-diaminopimelic acid (DAP) to l-lysine. The lysA gene of M. tuberculosis H37Rv has been established as essential for bacterial survival in immunocompromised mice, demonstrating that de novo biosynthesis of lysine is essential for in vivo viability. Drugs targeted against DAPDC could be efficient anti-tuberculosis drugs, and the three-dimensional structure of DAPDC from M. tuberculosis complexed with reaction product lysine and the ternary complex with PLP and lysine in the active site has been determined. The first structure of a DAPDC confirms its classification as a fold type III PLP-dependent enzyme. The structure shows a stable 2-fold dimer in head-to-tail arrangement of a triose-phosphate isomerase (TIM) barrel-like alpha/beta domain and a C-terminal beta sheet domain, similar to the ornithine decarboxylase (ODC) fold family. PLP is covalently bound via an internal aldimine, and residues from both domains and both subunits contribute to the binding pocket. Comparison of the structure with eukaryotic ODCs, in particular with a di-fluoromethyl ornithine (DMFO)-bound ODC from Trypanosoma bruceii, indicates that corresponding DAP-analogues might be potential inhibitors for mycobacterial DAPDCs.     

A mammalian type I fatty acid synthase acyl carrier protein domain does not sequester acyl chains

The synthases that produce fatty acids in mammals (FASs) are arranged as large multidomain polypeptides. The growing fatty acid chain is bound covalently during chain elongation and reduction to the acyl carrier protein (ACP) domain that is then able to access each catalytic site. In this work we report the high-resolution nuclear magnetic resonance (NMR) solution structure of the isolated rat fatty acid synthase apoACP domain. The final ensemble of NMR structures and backbone (15)N relaxation studies show that apoACP adopts a single, well defined fold. On conversion to the holo form, several small chemical shift changes are observed on the ACP for residues surrounding the phosphopantetheine attachment site (as monitored by backbone (1)H-(15)N correlation experiments). However, there are negligible chemical shift changes when the holo form is modified to either the hexanoyl or palmitoyl forms. For further NMR analysis, a (13)C,(15)N-labeled hexanoyl-ACP sample was prepared and full chemical shift assignments completed. Analysis of two-dimensional F(2)-filtered and three-dimensional (13)C-edited nuclear Overhauser effect spectroscopy experiments revealed no detectable NOEs to the acyl chain. These experiments demonstrate that unlike other FAS ACPs studied, this Type I ACP does not sequester a covalently linked acyl moiety, although transient interactions cannot be ruled out. This is an important mechanistic difference between the ACPs from Type I and Type II FASs and may be significant for the modulation and regulation of these important mega-synthases.     

Glutamate-41 of Vibrio harveyi acyl carrier protein is essential for fatty acid synthase but not acyl-ACP synthetase activity

Bacterial acyl carrier protein (ACP) is a small, acidic, and highly conserved protein that supplies acyl groups for biosynthesis of a variety of lipid products. Recent modelling studies predict that residues primarily in helix II of Escherichia coli ACP (Glu-41, Ala-45) are involved in its interaction with the condensing enzyme FabH of fatty acid synthase. Using recombinant Vibrio harveyi ACP as a template for site-directed mutagenesis, we have shown that an acidic residue at position 41 is essential for V. harveyi fatty acid synthase (but not acyl-ACP synthetase) activity. In contrast, various replacements of Ala-45 were tolerated by both enzymes. None of the mutations introduced dramatic structural changes based on circular dichroism and native gel electrophoresis. These results confirm that Glu-41 of ACP is a critical residue for fatty acid synthase, but not for all enzymes that utilize ACP as a substrate.     

The 1.3-Angstrom-resolution crystal structure of beta-ketoacyl-acyl carrier protein synthase II from Streptococcus pneumoniae

The beta-ketoacyl-acyl carrier protein synthases are members of the thiolase superfamily and are key regulators of bacterial fatty acid synthesis. As essential components of the bacterial lipid metabolic pathway, they are an attractive target for antibacterial drug discovery. We have determined the 1.3 A resolution crystal structure of the beta-ketoacyl-acyl carrier protein synthase II (FabF) from the pathogenic organism Streptococcus pneumoniae. The protein adopts a duplicated betaalphabetaalphabetaalphabetabeta fold, which is characteristic of the thiolase superfamily. The two-fold pseudosymmetry is broken by the presence of distinct insertions in the two halves of the protein. These insertions have evolved to bind the specific substrates of this particular member of the thiolase superfamily. Docking of the pantetheine moiety of the substrate identifies the loop regions involved in substrate binding and indicates roles for specific, conserved residues in the substrate binding tunnel. The active site triad of this superfamily is present in spFabF as His 303, His 337, and Cys 164. Near the active site is an ion pair, Glu 346 and Lys 332, that is conserved in the condensing enzymes but is unusual in our structure in being stabilized by an Mg(2+) ion which interacts with Glu 346. The active site histidines interact asymmetrically with Lys 332, whose positive charge is closer to His 303, and we propose a specific role for the lysine in polarizing the imidazole ring of this histidine. This asymmetry suggests that the two histidines have unequal roles in catalysis and provides new insights into the catalytic mechanisms of these enzymes.     

Complete amino acid sequence of chicken liver acyl carrier protein derived from the fatty acid synthase

The acyl carrier protein domain of the chicken liver fatty acid synthase has been isolated after tryptic treatment of the synthase. The isolated domain functions as an acceptor of acetyl and malonyl moieties in the synthase-catalyzed transfer of these groups from their coenzyme A esters and therefore indicates that the acyl carrier protein domain exists in the complex as a discrete entity. The amino acid sequence of the acyl carrier protein was derived from analyses of peptide fragments produced by cyanogen bromide cleavage and trypsin and Staphylococcus aureus V8 protease digestions of the molecule. The isolated acyl carrier protein domain consists of 89 amino acid residues and has a calculated molecular weight of 10,127. The protein contains the phosphopantetheine group attached to the serine residue at position 38. The isolated acyl carrier protein peptide shows some sequence homology with the acyl carrier protein of Escherichia coli, particularly in the vicinity of the site of phosphopantetheine attachment, and shows extensive sequence homology with the acyl carrier protein from the uropygial gland of goose.     

Malonyl-CoA: acyl carrier protein transacylase from Helicobacter pylori: Crystal structure and its interaction with acyl carrier protein

Malonyl-CoA: acyl carrier protein transacylase (MCAT) is a critical enzyme responsible for the transfer of the malonyl moiety to holo-acyl carrier protein (ACP) forming the malonyl-ACP intermediates in the initiation step of type II fatty acid synthesis (FAS II) in bacteria. MCAT has been considered as an attractive drug target in the discovery of antibacterial agents. In this study, the crystal structure of MCAT from Helicobacter pylori (Hp) at 2.5 angstroms resolution is reported, and the interaction of HpMCAT with HpACP is extensively investigated by using computational docking, GST-pull-down, and surface plasmon resonance (SPR) technology-based assays. The crystal structure results reveal that HpMCAT has a compact folding composed of a large subdomain with a similar core as in alpha/beta hydrolases, and a similar ferredoxin-like small subdomain as in acylphosphatases. The docking result suggests two positively charged areas near the entrance of the active site of HpMCAT as the ACP-binding region. Binding assay research shows that HpMCAT demonstrates a moderately binding ability against HpACP. The solved 3D structure of HpMCAT is expected to supply useful information for the structure-based discovery of novel inhibitors against MCAT, and the quantitative study of HpMCAT interaction with HpACP is hoped to give helpful hints in the understanding of the detailed catalytic mechanisms for HpMCAT.     

Acetoacetyl-acyl carrier protein synthase, a potential regulator of fatty acid biosynthesis in bacteria

The first condensation reaction in the fatty acid biosynthetic pathway in Escherichia coli was rate-limiting as judged by analysis of the relative pool sizes of acyl carrier protein (ACP) thioester intermediates in vivo. Comparable concentrations of acetyl-ACP, malonyl-ACP, and nonesterified ACP were present during logarithmic growth, whereas long-chain acyl-ACP comprised a minor fraction of the total ACP pool. The antibiotic cerulenin was used to irreversibly inhibit both beta-ketoacyl-ACP synthases I and II. However, acyl-ACP formation in vivo was not blocked by this antibiotic, and short-chain (4-8-carbon) acyl-ACPs increased to 60% of the total ACP pool in cerulenin-treated cells. These data suggested that existence of a cerulenin-resistant condensing enzyme that was capable of catalyzing the initial steps in chain elongation. A unique enzymatic activity, acetoacetyl-ACP synthase, that specifically catalyzed the condensation of malonyl-ACP and acetyl-ACP was detected in E. coli cell extracts. Acetoacetyl-ACP synthase activity was not inhibited by cerulenin and was present in extracts prepared from a double mutant harboring genetic lesions in beta-ketoacyl-ACP synthases I and II (fabB20 fabF3). These data point to the condensation of malonyl-ACP and acetyl-ACP as the rate-controlling reaction in fatty acid biosynthesis and implicate acetoacetyl-ACP synthase as the pacemaker of fatty acid production in organisms and organelles that possess dissociated (Type II) fatty acid synthase systems.     

Inactivation mechanism of the beta-ketoacyl-[acyl carrier protein] reductase of bacterial type-II fatty acid synthase by epigallocatechin gallate.

Epigallocatechin gallate (EGCG), a major compound from green tea, reversibly inhibits beta-ketoacyl-[acyl carrier protein] reductase (FabG) from Escherichia coli. In this study, we found that EGCG exhibited an atypical time-dependent inhibition of FabG, which possibly resulted from the EGCG-induced aggregation of FabG. It was observed that FabG inactivation and aggregation occurred nearly simultaneously, with a lag time that decreased with increasing EGCG concentration. These results suggest that some chemical reactions, required for aggregation and inactivation, occurred during the lag time. Since EGC was detected by HPLC after the incubation of EGCG with FabG, EGCG probably covalently modified FabG. These further results showed that 1 tetramer of FabG must be modified by several, possibly 4, EGCG molecules before the formation of FabG aggregates. FabG aggregation was a first-order reaction independent of protein concentration. Due to an initial lag time, the first-order rate of aggregation gradually increased, reaching a maximal and constant value. The effect of increasing concentration of EGCG on the first-order rate constant for aggregation indicated that EGCG bound to FabG by affinity labeling. Based on the results, we propose a mechanism for the interaction of EGCG with FabG:EGCG first binds reversibly to each subunit of FabG, followed by covalent modification and then aggregation of the 4 EGCG-modified subunits.     

Isoforms of acyl carrier protein involved in seed-specific fatty acid synthesis

Seeds of coriandrum sativum (coriander) and Thunbergia alata (black-eyed Susan vine) produce unusual monoenoic fatty acids which constitute over 80% of the total fatty acids of the seed oil. The initial step in the formation of these fatty acids is the desaturation of palmitoyl-ACP (acyl carrier protein) at the delta(4) or delta(6) positions to produce delta(4)-hexadecenoic acid (16:1(delta(4)) or delta(6)-hexadecenoic acid (16:1(delta(6)), respectively. The involvement of specific forms of ACP in the production of these novel monoenoic fatty acids was studied. ACPs were partially purified from endosperm of coriander and T. alata and used to generate 3H- and 14C-labelled palmitoyl-ACP substrates. In competition assays with labelled palmitoyl-ACP prepared from spinach (Spinacia oleracea), delta(4)-acyl-ACP desaturase activity was two- to threefold higher with coriander ACP than with spinach ACP. Similarly, the T. alata delta(6) desaturase favoured T. alata ACP over spinach ACP. A cDNA clone, Cs-ACP-1, encoding ACP was isolated from a coriander endosperm cDNA library. Cs-ACP-1 mRNA was predominantly expressed in endosperm rather than leaves. The Cs-ACP-1 mature protein was expressed in E. coli and comigrated on SDS-PAGE with the most abundant ACP expressed in endosperm tissues. In in vitro delta(4)-palmitoyl-ACP desaturase assays, the Cs-ACP-1 expressed from E. coli was four- and 10-fold more active than spinach ACP or E. coli ACP, respectively, in the synthesis of delta(4)-hexadecenoic acid from palmitoyl-ACP. In contrast, delta(9)-stearoyl-ACP desaturase activity from coriander endosperm did not discriminate strongly between different ACP species. These results indicate that individual ACP isoforms are specifically involved in the biosynthesis of unusual seed fatty acids and further suggest that expression of multiple ACP isoforms may participate in determining the products of fatty acid biosynthesis.     

Engineered fatty acid biosynthesis in Streptomyces by altered catalytic function of beta-ketoacyl-acyl carrier protein synthase III

The Streptomyces glaucescens beta-ketoacyl-acyl carrier protein (ACP) synthase III (KASIII) initiates straight- and branched-chain fatty acid biosynthesis by catalyzing the decarboxylative condensation of malonyl-ACP with different acyl-coenzyme A (CoA) primers. This KASIII has one cysteine residue, which is critical for forming an acyl-enzyme intermediate in the first step of the process. Three mutants (Cys122Ala, Cys122Ser, Cys122Gln) were created by site-directed mutagenesis. Plasmid-based expression of these mutants in S. glaucescens resulted in strains which generated 75 (Cys122Ala) to 500% (Cys122Gln) more straight-chain fatty acids (SCFA) than the corresponding wild-type strain. In contrast, plasmid-based expression of wild-type KASIII had no effect on fatty acid profiles. These observations are attributed to an uncoupling of the condensation and decarboxylation activities in these mutants (malonyl-ACP is thus converted to acetyl-ACP, a SCFA precursor). Incorporation experiments with perdeuterated acetic acid demonstrated that 9% of the palmitate pool of the wild-type strain was generated from an intact D(3) acetyl-CoA starter unit, compared to 3% in a strain expressing the Cys122Gln KASIII. These observations support the intermediacy of malonyl-ACP in generating the SCFA precursor in a strain expressing this mutant. To study malonyl-ACP decarboxylase activity in vitro, the KASIII mutants were expressed and purified as His-tagged proteins in Escherichia coli and assayed. In the absence of the acyl-CoA substrate the Cys122Gln mutant and wild-type KASIII were shown to have comparable decarboxylase activities in vitro. The Cys122Ala mutant exhibited higher activity. This activity was inhibited for all enzymes by the presence of high concentrations of isobutyryl-CoA (>100 microM), a branched-chain fatty acid biosynthetic precursor. Under these conditions the mutant enzymes had no activity, while the wild-type enzyme functioned as a ketoacyl synthase. These observations indicate the likely upper and lower limits of isobutyryl-CoA and related acyl-CoA concentrations within S. glaucescens.     

A small, discrete acyl carrier protein is involved in de novo fatty acid biosynthesis in Streptomyces erythraeus

A heat-stable factor, required for de novo synthesis of fatty acids in the erythromycin-producing organism Streptomyces erythraeus, has been purified to homogeneity and identified as an acyl carrier protein (ACP). We conclude that, contrary to previous belief, fatty acid synthase in S. erythraeus more closely resembles the dissociable complex of E. coli than the tightly associated, multifunctional enzyme complex found in the related actinomycete Mycobacterium smegmatis.     

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.     

FeeM, an N-acyl amino acid synthase from an uncultured soil microbe: structure, mechanism, and acyl carrier protein binding

Attempts to access antibiotics by capturing biosynthetic genes and pathways directly from environmental DNA, which is overwhelmingly derived from uncultured bacteria, have revealed a large and previously unknown family of N-acyl amino acid synthases (NASs). The structure of the NAS FeeM reveals structural similarity to the GCN5-related N-acyl transferases and acylhomoserine lactone synthases. The overall structure has a central beta sheet with alpha helices on both sides. A bound product at a cleft in the beta sheet identifies the active site and the structural basis for catalysis, and sequence conservation in this region indicates a bias for recognition over speed. FeeM interacts with an acyl carrier protein (FeeL), and the structure, mutagenesis, and enzymatic measurements reveal that a small hydrophobic pocket in alpha helix 5 dominates binding of FeeM to FeeL. The structural and mechanistic analyses suggest that the products of FeeM could be bacterial signaling agents.