Quantification of Acyl-Acyl Carrier Proteins for Fatty Acid Synthesis Using LC-MS/MS.

The fatty acid biosynthetic cycle is predicated on an acyl carrier protein (ACP) scaffold where two carbon acetyl groups are added in a chain elongation process through a series of repeated enzymatic steps. The chain extension is terminated by hydrolysis with a thioesterase or direct transfer of the acyl group to a glycerophospholipid by an acyltransferase. Methods for analysis of the concentrations of acyl chains attached to ACPs are lacking but would be informative for studies in lipid metabolism. We describe a method to profile and quantify the levels of acyl-ACPs in plants, bacteria and mitochondria of animals and fungi that represent Type II fatty acid biosynthetic systems. ACPs of Type II systems have a highly conserved Asp-Ser-Leu-Asp (DSLD) amino acid sequence at the attachment site for 4'-phosphopantetheinyl arm carrying the acyl chain. Three amino acids of the conserved sequence can be cleaved away from the remainder of the protein using an aspartyl protease. Thus, partially purified protein can be enzymatically hydrolyzed to produce an acyl chain linked to a tripeptide via the 4'-phosphopantetheinyl group. After ionization and fragmentation, the corresponding fragment ion is detected by a triple quadrupole mass spectrometer using a multiple reaction monitoring method. 15N isotopically labeled acyl-ACPs generated in high amounts are used with an isotope dilution strategy to quantify the absolute levels of each acyl group attached to the acyl carrier protein scaffold.

A Priming Cassette Generates Hydroxylated Acyl Starter Units in Mupirocin and Thiomarinol Biosynthesis.

Mupirocin, a commercially available antibiotic produced by Pseudomonas fluorescens NCIMB 10586, and thiomarinol, isolated from the marine bacterium Pseudoalteromonas sp. SANK 73390, both consist of a polyketide-derived monic acid homologue esterified with either 9-hydroxynonanoic acid (mupirocin, 9HN) or 8-hydroxyoctanoic acid (thiomarinol, 8HO). The mechanisms of formation of these deceptively simple 9HN and 8HO fatty acid moieties in mup and tml, respectively, remain unresolved. To define starter unit generation, the purified mupirocin proteins MupQ, MupS, and MacpD and their thiomarinol equivalents (TmlQ, TmlS and TacpD) have been expressed and shown to convert malonyl coenzyme A (CoA) and succinyl CoA to 3-hydroxypropionoyl (3-HP) or 4-hydroxybutyryl (4-HB) fatty acid starter units, respectively, via the MupQ/TmlQ catalyzed generation of an unusual bis-CoA/acyl carrier protein (ACP) thioester, followed by MupS/TmlS catalyzed reduction. Mix and match experiments show MupQ/TmlQ to be highly selective for the correct CoA. MacpD/TacpD were interchangeable but alternate trans-acting ACPs from the mupirocin pathway (MacpA/TacpA) or a heterologous ACP (BatA) were nonfunctional. MupS and TmlS selectivity was more varied, and these reductases differed in their substrate and ACP selectivity. The solution structure of MacpD determined by NMR revealed a C-terminal extension with partial helical character that has been shown to be important for maintaining high titers of mupirocin. We generated a truncated MacpD construct, MacpD_T, which lacks this C-terminal extension but retains an ability to generate 3-HP with MupS and MupQ, suggesting further downstream roles in protein-protein interactions for this region of the ACP.

Rv0100, a proposed acyl carrier protein in Mycobacterium tuberculosis: expression, purification and crystallization.

Acyl carrier proteins (ACPs) are important components in fatty-acid biosynthesis in prokaryotes. Rv0100 is predicted to be an essential ACP in Mycobacterium tuberculosis, the pathogen that is the causative agent of tuberculosis, and therefore has the potential to be a novel antituberculosis drug target. Here, the successful cloning and purification of Rv0100 using Mycobacterium smegmatis as a host is reported. Crystals of the purified protein were obtained that diffracted to a resolution of 1.9 Å. Overall, this work lays the foundation for the future pursuit of drug discovery and development against this potentially novel drug target.

Recognition of (2S)-aminomalonyl-acyl carrier protein (ACP) and (2R)-hydroxymalonyl-ACP by acyltransferases in zwittermicin A biosynthesis.

Polyketide synthases elongate a polyketide backbone by condensing carboxylic acid precursors that are thioesterified to either coenzyme A or an acyl carrier protein (ACP). Two of the three known ACP-linked extender units, (2S)-aminomalonyl-ACP and (2R)-hydroxymalonyl-ACP, are found in the biosynthesis of the agriculturally important antibiotic zwittermicin A. We previously reconstituted the formation of (2S)-aminomalonyl-ACP and (2R)-hydroxymalonyl-ACP from the primary metabolites l-serine and 1,3-bisphospho-d-glycerate. In this report, we characterize the two acyltransferases involved in the specific transfer of the (2S)-aminomalonyl and (2R)-hydroxymalonyl moieties from the ACPs associated with extender unit formation to the ACPs integrated into the polyketide synthase. This work establishes which acyltransferase recognizes each extender unit and also provides insight into the substrate selectivity of these enzymes. These are important step toward harnessing these rare polyketide synthase extender units for combinatorial biosynthesis.

Expression, purification and characterization of the acyl carrier protein phosphodiesterase from Pseudomonas Aeruginosa.

Acyl carrier protein phosphodiesterases (AcpH) are the only enzymes known to remove the 4'-phosphopantetheinyl moiety from holo acyl carrier proteins (ACP), which are a large family of proteins essential for the biosynthesis of lipid and other cellular metabolites. Here we report that the AcpH (paAcpH) from Pseudomonas aeruginosa can be overexpressed in Escherichia coli as a soluble and stable protein after optimization of the expression and purification conditions. This marks an improvement from the aggregation-prone E. coli AcpH that could only be obtained by refolding the polypeptide obtained from the inclusion body. With the soluble recombinant protein, we found that PaAcpH exhibits preferred substrate specificity towards the ACPs from the fatty acid synthesis pathway among eight carrier proteins. We further showed that PaAcpH hydrolyzes and releases the 4'-phosphopantetheinyl group-linked products from a multidomain polyketide synthase, demonstrating that the enzyme is fully capable of hydrolyzing acylated ACP substrates.

In vivo modification of native carrier protein domains.

Carrier proteins are central to the biosynthesis of primary and secondary metabolites in all organisms. Here we describe metabolic labeling and manipulation of native acyl carrier proteins in both type I and II fatty acid synthases. By utilizing natural promiscuity in the CoA biosynthetic pathway in combination with synthetic pantetheine analogues, we demonstrate metabolic labeling of endogenous carrier proteins with reporter tags in Gram-positive and Gram-negative bacteria and in a human carcinoma cell line. The highly specific nature of the post-translational modification that was utilized for tagging allows for simple visualization of labeled carrier proteins, either by direct fluorescence imaging or after chemical conjugation to a fluorescent reporter. In addition, we demonstrate the utility of this approach for the isolation and enrichment of carrier proteins by affinity purification. Finally, we use these techniques to identify a carrier protein from an unsequenced organism, a finding that validates this proteomic approach to natural product biosynthetic enzyme discovery.

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.

Expression and characterization of polyketide synthase module involved in the late step of cephabacin biosynthesis from Lysobacter lactamgenus.

The cephabacins produced by Lysobacter lactamgenus are beta-lactam antibiotics composed of a cephem nucleus, an acetate residue, and an oligopeptide side chain. In order to understand the precise implication of the polyketide synthase (PKS) module in the biosynthesis of cephabacin, the genes for its core domains, beta-ketoacyl synthase (KS), acyltransferase (AT), and acyl carrier protein (ACP), were amplified and cloned into the pET-32b(+) expression vector. The sfp gene encoding a protein that can modify apo-ACP to its active holo-form was also amplified. The recombinant KS, AT, apo-ACP, and Sfp overproduced in the form of His6-tagged fusion proteins in E. coli BL21(DE3) were purified by nickel-affinity chromatography. Formation of stable peptidyl-S-KS was observed by in vitro acylation of the KS domain with the substrate [L-Ala-L-Ala-LAla- L-3H-Arg] tetrapeptide-S-N-acetylcysteamine, which is the evidence for the selective recognition of tetrapeptide produced by nonribosomal peptide synthetase (NRPS) in the NRPS/ PKS hybrid. In order to confirm whether malonyl CoA is the extender unit for acetylation of the peptidyl moiety, the AT domain, ACP domain, and Sfp protein were treated with 14C-malonyl-CoA. The results clearly show that the AT domain is able to recognize the extender unit and decarboxylatively acetylated for the elongation of the tetrapeptide. However, the transfer of the activated acetyl group to the ACP domain was not observed, probably attributed to the improper capability of Sfp to activate apo-ACP to the holo-ACP form.

An orthogonal purification strategy for isolating crosslinked domains of modular synthases.

Chemo-enzymatic methods for covalently crosslinking carrier proteins with partner enzymes within modular synthases hold promise for elucidating and engineering metabolic pathways. Our efforts to crystallize the ACP-KS complexes of fatty acid synthases have been complicated by difficulties in the purification of the crosslinked complex from the other proteins in the reaction. Here we present a solution that employs an orthogonal purification strategy to achieve the quantity and level of purity necessary for further studies of this complex.

Helicobacter pylori acyl carrier protein: expression, purification, and its interaction with beta-hydroxyacyl-ACP dehydratase.

Acyl carrier protein (ACP) is an essential component in the type II fatty acid biosynthesis (FAS II) process and is responsible for the acyl group transfer within a series of related enzymes. In this work, the ACP from Helicobacter pylori strain SS1 was cloned and the gene sequence of Hpacp was deposited in the GenBank database (Accession No.: AY904356). Two forms of HpACP (apo, holo) were successfully purified and characterized. The thermal stability of these two forms was quantitatively investigated by CD spectral analyses. The results revealed that the holo-HpACP was more stable than apo-HpACP according to the transition midpoint temperature(Tm). Moreover, the interaction of HpACP with the related enzyme (beta-hydroxyacyl-ACP dehydratase, HpFabZ) was determined by GST-pull down assay and surface plasmon resonance (SPR) technique in vitro, the results showed that HpACP displays a strong binding affinity to HpFabZ (KD=1.2 x 10(-8)M). This current work is hoped to supply useful information for better understanding the ACP features of Helicobacter pylori SS1 strain.

Hydroxymalonyl-acyl carrier protein (ACP) and aminomalonyl-ACP are two additional type I polyketide synthase extender units.

Combinatorial biosynthesis of type I polyketide synthases is a promising approach for the generation of new structural derivatives of polyketide-containing natural products. A target of this approach has been to change the extender units incorporated into a polyketide backbone to alter the structure and activity of the natural product. One limitation to these efforts is that only four extender units were known: malonyl-CoA, methylmalonyl-CoA, ethylmalonyl-CoA, and methoxymalonyl-acyl carrier protein (ACP). The chemical attributes of these extender units are quite similar, with the exception of the potential hydrogen bonding interactions by the oxygen of the methoxy moiety. Furthermore, the incorporated extender units are not easily modified by using simple chemical approaches when combinatorial biosynthesis is coupled to semisynthetic chemistry. We recently proposed the existence of two additional extender units, hydroxymalonyl-ACP and aminomalonyl-ACP, involved in the biosynthesis of zwittermicin A. These extender units offer unique possibilities for combinatorial biosynthesis and semisynthetic chemistry because of the introduction of free hydroxyl and amino moieties into a polyketide structure. Here, we present the biochemical and mass spectral evidence for the formation of these extender units. This evidence shows the formation of ACP-linked extender units for polyketide synthesis. Interestingly, aminomalonyl-ACP formation involves enzymology typically found in nonribosomal peptide synthesis.

Characterization and site-directed mutagenesis of the putative novel acyl carrier protein Rv0033 and Rv1344 from Mycobacterium tuberculosis.

Mycolic acids are generated in Mycobacterium tuberculosis as a result of the interaction of two fatty acid biosynthetic systems: type I fatty acid synthase (FAS) and type II fatty acid synthase. Acyl carrier protein (ACP) is a small, acidic protein in type II FAS systems. It plays a central role in mycolic acid biosynthesis by transferring the acyl groups from one enzyme to another for the completion of the fatty acid synthesis cycle. The nature of the proper recognition between ACPs and its many interactive proteins is not understood. Here, we report the over-expression, purification, and characterization of two putative ACPs: Rv0033 and Rv1344 in M. tuberculosis. In order to study the role of the conserved residues and the conformation of whole protein, some site-directed mutations of recombinant Acp1344 were made and the 3D structure of Acp1344 was modeled.

Gene cloning, expression and functional characterization of an acyl carrier protein AcpV from Vibrio anguillarum.

Acyl carrier protein (ACP) is a small acidic protein that acts as an essential cofactor in many biosynthetic pathways depending on acyl transfer reactions. In this work, a Vibrio anguillarum ACP encoding gene, acpV, was first cloned from the chromosome of a virulent V. anguillarum strain MVM425. acpV was over-expressed in Escherichia coli and the resultant protein AcpV was purified. The purified AcpV was incubated with purified phosphopantetheinyl transferase (PPtase) in the presence of CoA to assay the 4'-phosphopantetheinylation of AcpV in vitro; and on the other hand, the acpV gene was co-expressed with PPtase-encoding gene in E. coli to examine the 4'-phosphopantetheinylation of AcpV in vivo. Our results suggested that acpV encoded a functional ACP of V. anguillarum, which can be 4'-phosphopantetheinylated well by AcpS-type PPtase (E. coli AcpS) both in vitro and in vivo, but cannot serve as a good substrate for Sfp-type PPtase (V. anguillarum AngD).

Genomic structures and characterization of the 5'-flanking regions of acyl carrier protein and Delta4-palmitoyl-ACP desaturase genes from Coriandrum sativum.

The seed-specific or seed-predominant promoters of acyl carrier protein (Cs-ACP1) and Delta4-palmitoyl-acyl carrier protein desaturase (Cs-4PAD) genes, which are involved in the biosynthesis of petroselinic acid, were isolated from coriander (Coriandrum sativum) and analyzed in coriander endosperms and transgenic Arabidopsis. The expression of Cs-ACP1 and Cs-4PAD genes was coordinately regulated during seed development.

Nonorganellar acyl carrier protein from oleaginous yeast is a homologue of ribosomal protein P2.

Acyl carrier protein (ACP) is responsible for carrying the growing fatty acid chain from one enzyme active site to the next during fatty acid biosynthesis. Here we report the identification, purification, immunocytochemical localization, and cloning of ACP from the oleaginous yeast, Rhodotorula glutinis. The soluble fraction of this organism can synthesize triacylglycerol and is able to accept the acyl group from acyl-ACP for the synthesis. The ACP, cloned from the system, showed a significant similarity with ribosomal protein P2. Expression and characterization of the recombinant protein showed that the ACP was acylated in vitro. The recombinant protein was post-translationally modified, since it was observed in [14C]beta-alanine labeling and matrix-assisted laser desorption mass spectroscopic analysis. Site-directed mutants were generated to identify a serine residue responsible for phosphopantetheinylation and found that mutation of serine 59 to alanine abrogated the fatty acylation ability of the protein. These results demonstrate that a novel modification of ribosomal protein P2 allows it to act as an acyl carrier protein and participate in acylation reactions.

New partners of acyl carrier protein detected in Escherichia coli by tandem affinity purification.

We report the first use of tandem affinity purification (TAP) in a prokaryote to purify native protein complexes, and demonstrate its reliability and power. We purified the acyl carrier protein (ACP) of Escherichia coli, a protein involved in a myriad of metabolic pathways. Besides the identification of several known partners of ACP, we rediscovered ACP/MukB and ACP/IscS interactions already detected but previously disregarded as due to contamination. Here, we demonstrate the specificity of these interactions and characterize them. This suggests that ACP is involved in additional previously unsuspected pathways. Furthermore, this study shows how the TAP method can be simply used in prokaryotes such as E. coli to identify new partners in protein-protein interactions under physiological conditions and thereby uncover novel protein functions.

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.

Identification of a key residue in the conformational stability of acyl carrier protein.

Conformational flexibility of acyl carrier protein (ACP) is important for its ability to interact with multiple enzymes in bacterial fatty acid metabolism. We have recently shown that, unlike the prototypical ACP from Escherichia coli, the more acidic Vibrio harveyi ACP is largely unfolded at physiological pH. Mutations D18K, A75H and A75H/D18K were made in recombinant V. harveyi ACP (rACP) to determine the importance of basic residues Lys-18 and His-75 in maintaining the native conformation of E. coli ACP. Both D18K and A75H ACPs were fatty acylated by acyl-ACP synthetase, showing that neither mutation grossly alters tertiary structure. Circular dichroism (CD) indicated that rACP refolded upon addition of MgCl(2) at 100-fold lower concentrations (<1 mM) than KCl, suggesting that divalent cations stabilize rACP by interaction at specific sites. Surprisingly, mutants A75H and A75H/D18K exhibited native-like conformation in the absence of MgCl(2), while the D18K mutant was comparable to rACP. Moreover, the alpha-helical content of A75H, A75H/D18K and E. coli ACPs was more sensitive than that of rACP or D18K ACP to modification by the histidine-selective reagent diethylpyrocarbonate. Together, these results suggest that the partial positive charge of His-75 may be important in maintaining the conformational stability of E. coli ACP at a neutral pH.

Fast characterization of intact proteins using a high-throughput eight-channel parallel liquid chromatography/mass spectrometry system.

The preparation of protein substrates requires that a large number of chromatographic fractions be analyzed for the presence of reactants, products and by-products. Analyses using linear matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) or single column liquid chromatography/mass spectrometry (LC/MS) have been inadequate because of mass resolution or throughput. Therefore, a high-throughput method employing an eight-channel parallel reverse-phase LC/MS system was developed. This system is capable of screening fractions from preparative ion-exchange chromatography with the required mass accuracy and throughput so that the protein purification process can be monitored in a relatively short period of time. As an example, the purification and analysis of an acylated protein with a molecular weight of 8.9 kDa is described and the detection of a contaminating by-product that differs in size by less than 20 Da is demonstrated. Using the current instrumentation and approach, it is practical to analyze 50 protein-containing fractions from column chromatography in less than 1 hour using parallel LC/MS.

Purification, priming, and catalytic acylation of carrier protein domains in the polyketide synthase and nonribosomal peptidyl synthetase modules of the HMWP1 subunit of yersiniabactin synthetase.

The 207-kDa polyketide synthase (PKS) module (residues 1-1895) and the 143-kDa nonribosomal peptidyl synthetase (NRPS) module (1896-3163) of the 350-kDa HMWP1 subunit of yersiniabactin synthetase have been expressed in and purified from Escherichia coli in soluble forms to characterize the acyl carrier protein (ACP) domain of the PKS module and the homologous peptidyl carrier protein (PCP(3)) domain of the NRPS module. The apo-ACP and PCP domains could be selectively posttranslationally primed by the E. coli ACPS and EntD phosphopantetheinyl transferases (PPTases), respectively, whereas the Bacillus subtilis PPTase Sfp primed both carrier protein domains in vitro or during in vivo coexpression. The holo-NRPS module but not the holo-PKS module was then selectively aminoacylated with cysteine by the adenylation domain embedded in the HMWP2 subunit of yersiniabactin synthetase, acting in trans. When the acyltransferase (AT) domain of HMWP1 was analyzed for its ability to malonylate the holo carrier protein domains, in cis acylation was first detected. Then, in trans malonylation of the excised holo-ACP or holo-PCP(3)-TE fragments by HMWP1 showed both were malonylated with a 3:1 catalytic efficiency ratio, showing a promiscuity to the AT domain.