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Acyl carrier proteins (ACPs) are required for the transfer of acyl intermediates during fatty acid and polyketide syntheses. InSinorhizobium meliloti1021 there are five known ACPs: AcpP, NodF, AcpXL, the ACP domain in RkpA and SMb20651. The genome sequence ofS. meliloti1021 also reveals the ORF SMc01553, annotated as a putative ACP.smc01553is part of a 6.6 kb DNA region that is duplicated in the chromosome and in the pSymb plasmid, the result of a recent duplication event. SMc01553 overexpressed inEscherichia coliwas labelledin vivowith [3H]β-alanine, a biosynthetic building block of the 4′-phosphopantetheine prosthetic group of ACPs. The purified SMc01553 was modified with 4′-phosphopantetheine in the presence ofS. melilotiholo-ACP synthase, and this modification resulted in a major conformational change of the protein structure, since the holo-form runs faster in native PAGE than the apo-form. SMc01553 could not be loaded with a malonyl group by malonyl-CoA-ACP transacylase fromS. meliloti. Using RT-PCR we could show the presence of mRNA for SMc01553 and of the duplicated ORF SMb22007 in cultures ofS. meliloti. However, a mutant in which the two duplicated regions were deleted did not show any different phenotype with respect to the wild-type in the free-living or symbiotic lifestyle.

Polyketides are a class of natural products with diverse structures and biological activities. The structural variability of aromatic products of fungal nonreducing, multidomain iterative polyketide synthases (NR-PKS group of IPKSs) results from regiospecific cyclizations of reactive poly-beta-keto intermediates. How poly-beta-keto species are synthesized and stabilized, how their chain lengths are determined, and, in particular, how specific cyclization patterns are controlled have been largely inaccessible and functionally unknown until recently. A product template (PT) domain is responsible for controlling specific aldol cyclization and aromatization of these mature polyketide precursors, but the mechanistic basis is unknown. Here we present the 1.8 A crystal structure and mutational studies of a dissected PT monodomain from PksA, the NR-PKS that initiates the biosynthesis of the potent hepatocarcinogen aflatoxin B(1) in Aspergillus parasiticus. Despite having minimal sequence similarity to known enzymes, the structure displays a distinct 'double hot dog' (DHD) fold. Co-crystal structures with palmitate or a bicyclic substrate mimic illustrate that PT can bind both linear and bicyclic polyketides. Docking and mutagenesis studies reveal residues important for substrate binding and catalysis, and identify a phosphopantetheine localization channel and a deep two-part interior binding pocket and reaction chamber. Sequence similarity and extensive conservation of active site residues in PT domains suggest that the mechanistic insights gleaned from these studies will prove general for this class of IPKSs, and lay a foundation for defining the molecular rules controlling NR-PKS cyclization specificity.

The type II thioesterase EntH is a hotdog fold protein required for optimal nonribosomal biosynthesis of enterobactin in Escherichia coli. Its proposed proofreading activity in the biosynthesis is confirmed by its efficient restoration of enterobactin synthesis blocked in vitro by analogs of the cognate precursor 2,3-dihydroxybenzoate. Steady-state kinetic studies show that EntH recognizes the phosphopantetheine group and the pattern of hydroxylation in the aryl moiety of its thioester substrates. Remarkably, it is able to distinguish aberrant intermediates from the normal one in the enterobactin assembly line by demonstrating at least 10-fold higher catalytic efficiency toward thioesters derived from aberrant aryl precursors without a para-hydroxyl group, such as salicylate. By structural comparison and site-directed mutagenesis, the thioesterase is found to possess an active site closely resembling that of the 4-hydroxybenzoyl-CoA thioesterase from Arthrobacter sp. strain SU and to involve an acidic residue (glutamate-63) as the catalytic base or nucleophile like all other hotdog thioesterases. In addition, the EntH specificities toward the substrate hydroxylation pattern are found to depend on the active-site histidine-54, threonine-64, serine-67, and methionine-68 with the selectivity significantly reduced or even reversed when they are individually replaced by alanine. These residues are likely responsible for differential interaction of the enzyme with the substrates which leads to distinction between the normal and aberrant precursors in the enterobactin assembly line. These results show that the type II thioesterase evolves its distinctive ability to recognize the aberrant intermediates from the versatile catalytic platform of hotdog proteins and suggests an active search mechanism for type II thioesterases in nonribosomal peptide synthesis.

Acyl carrier proteins (ACPs) are essential to both fatty acid synthase (FAS) and polyketide synthase (PKS) biosynthetic pathways, yet relatively little is known about how they function at a molecular level. Seven thiol ester and thiol ether derivatives of the actinorhodin (act) PKS ACP from Streptomyces coelicolor have been prepared and structurally characterised by NMR to gain insight into ACP-intermediate interactions. Holo ACP synthase has been used to prepare early-stage ACP intermediates of polyketide biosynthesis (holo ACP, acetyl ACP, and malonyl ACP) from the respective coenzyme A derivatives. A synthetic route to stabilised thiol ether ACPs was developed and applied to the preparation of stable 3-oxobutyl and 3,5-dioxohexyl ACP as diketide and triketide analogues. No interaction between the protein and the acyl phosphopantetheine moieties of acetyl, malonyl, or 3-oxobutyl ACP was detected. Analysis of (1)H-(15)N heteronuclear single quantum coherence and nuclear Overhauser enhancement spectroscopy spectra for the triketide ACP revealed exchange between a major ('Tri', 85%) and a minor protein conformer in which the polyketide interacts with the protein ('Tri(*)', 15%). Act ACP was also derivatised with butyryl, hexanoyl, and octanoyl groups. The corresponding NMR spectra showed large chemical shift perturbations centred on helices II and III, indicative of acyl chain binding and significant structural rearrangement. Unexpectedly, butyryl act ACP showed almost identical backbone (1)H-(15)N chemical shifts to Tri(*), suggesting comparable structural changes that might provide insight into the structurally uncharacterised polyketide bound form. Furthermore, butyryl ACP itself underwent slow conformational exchange with a second minor conformer (But(*)) with almost identical backbone chemical shifts to octanoyl act ACP. High-resolution NMR structures of these acylated forms revealed that act ACP was able to undergo dramatic conformational changes that exceed those seen in FAS ACPs. When compared to E. coli FAS ACP, the substrate binding pocket of the act PKS ACP has three specific amino acid substitutions (Thr39/Leu45, Ala68/Leu74, and Leu42/Thr48) that alter the size, shape, and location of this cavity. These conformational changes may play a role in protein-protein recognition and assist the binding of bulky polyketide intermediates.

Acyl carrier proteins (ACPs) are small acidic proteins that carry growing acyl chains during fatty acid or polyketide synthesis. In rhizobia, there are four different and well-characterized ACPs: AcpP, NodF, AcpXL and RkpF. The genome sequence ofSinorhizobium meliloti1021 reveals two additional ORFs that possibly encode additional ACPs. One of these,smb20651, is located on the plasmid pSymB as part of an operon. The genes of the operon encode a putative asparagine synthetase (AsnB), the predicted ACP (SMb20651), a putative long-chain fatty acyl-CoA ligase (SMb20650) and a putative ammonium-dependent NAD+synthetase (NadE1). When SMb20651 was overexpressed inEscherichia coli,[3H]β-alanine, a biosynthetic building block of 4′-phosphopantetheine, was incorporated into the proteinin vivo. The purified SMb20651 was modified with 4′-phosphopantetheine in the presence ofS. melilotiholo-ACP synthase (AcpS). Also, holo-SMb20651 was modifiedin vitrowith a malonyl group by malonyl CoA-ACP transacylase. InE. coli, coexpression of SMb20651 together with other proteins such as AcpS and SMb20650 led to the formation of additional forms of SMb20651. In this bacterium, acylation of SMb20651 with C12 : 0 or C18 : 0 fatty acids was detected, demonstrating that this protein is involved in fatty acid biosynthesis or transfer. Expression of SMb20651 was detected inS. melilotias holo-SMb20651 and acyl-SMb20651.

No data exist on the ability of thiolation domains from fungal non-ribosomal peptide synthetases to undergo 4'-phosphopantetheinylation, using either biotinylated or fluorescently labeled coenzyme A analogues, mediated by 4'-phosphopantetheinyl transferases (PPTase). Yet, this is a key requirement to confirm the amino acid recognition function, and coding potential, of either non-ribosomal peptide synthetases or recombinantly expressed regions of these enzymes (e.g., didomains or modules). Moreover, determination of 4'-phosphopantetheinylation activity remains cumbersome. Here, we demonstrate that a recombinant fungal PPTase catalyzes the solution-phase transfer of either biotin- or fluorescein-labeled 4'-phosphopantetheine region of coenzyme A to a fungal thiolation domain, which is either part of a non-ribosomal peptide synthetase didomain (72 kDa), derived from Aspergillus fumigatus, or fused to a non-native protein (glutathione s-transferase). Significantly, we demonstrate that this reaction can unexpectedly occur when the target protein (4.4 pmol) is immobilized on a solid surface. These findings (i) confirm that thiolation domains of fungal origin, in native or non-native configuration, can accept modified 4'-phosphopantetheine residues via PPTase-mediated labeling and (ii) illustrate a novel, high-throughput method to determine PPTase activity.

Acyl carrier protein (ACP) contains an essential sulfhydryl group in its phosphopantetheine prosthetic group. We have investigated the state of this sulfhydryl in developing and mature spinach seed (Spinacia oleracea). Seed extracts were separated on sodium dodecyl sulfate or native polyacrylamide gels, blotted to nitrocellulose, and probed with antibodies raised against spinach ACP-I. In extracts of mature seeds prepared with reducing agents, ACP-II migrated as a single major band, whereas extracts prepared without reducing agents gave two major bands. The additional band was identified as a conjugate of ACP-II to glutathione (ACP-S-S-G) on the basis of its sensitivity to reducing agents and its comigration with standards in both native and sodium dodecyl sulfate gel electrophoresis. In developing spinach seeds ACP-II exists primarily in its free sulfhydryl form or as acyl derivatives, with essentially no ACP-S-S-G present. During later stages of seed development, as seed water content declines, ACP-S-S-G accumulates to approximately 50% of the total ACP. Seed imbibition results in a rapid decline in ACP-S-S-G levels. The ACP-S-S-G:ACP-SH ratio of seeds during storage was found to be a function of seed water content and this could be manipulated by controlling the relative humidity under which the seeds were stored. We speculate that conjugation of ACP to glutathione protects the ACP from sulfhydryl oxidative damage in dry seeds.

Rhizobium species produce a protein product of the nodF gene that has a limited but recognizable homology to the well-characterized acyl carrier protein (ACP) of Escherichia coli. NodF functions together with NodE in generating a host-specific response to the plant host in the interchange of signals leading to the effective nodulation of roots (H.P. Spaink, J. Weinman, M.A. Djordjevic, C.A. Wijffelman, R.J.H. Okker, and B. J.J. Lugtenberg, EMBO J. 8:2811-2818, 1989; B. Scheres, C. van de Wiel, A. Zalensky, B. Horvath, H. Spaink, H. van Eck, F. Zwartkruis, A.M. Wolters, T. Gloudemans, A. van Kammen, and T. Bisseling, Cell 60:281-294, 1990). The nodFE region of Rhizobium leguminosarum has been cloned into a multicopy plasmid and has been shown in R. leguminosarum to code for a flavonoid-inducible protein that is effectively labeled by radioactive beta-alanine added to the growth medium. After purification, the labeled protein migrates as a single band with an apparent molecular weight of 5,000 during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, more rapidly than E. coli ACP. In contrast, in native gels the protein is resolved into two bands, both identified as NodF by analysis of the amino terminus and both migrating more slowly than E. coli ACP. Pulse-chase experiments with labeled beta-alanine suggested that the slower-moving band may be the precursor of the faster band. The NodF protein carries a 4'-phosphopantetheine as a prosthetic group. A NodF fusion protein under the control of the lac promoter is expressed in E. coli and is labeled with beta-alanine, indicating that it is recognized by the ACP synthase of E. coli. The ACP phosphodiesterase of E. coli, which catalyzes the release of phosphopantetheine from E. coli ACP, does not remove phosphopantetheine from NodF.

Recently a chloroplast holo-acyl carrier protein (holoACP) synthase activity was identified which attached the phosphopantetheine prosthetic group to acyl carrier protein, producing holoACP (Fernandez and Lamppa (1990) Plant Cell 2, 195-206). Here we show that the mature form of ACP (apoACP), after entry into the chloroplast and removal of the transit peptide, is a substrate for modification by the holoACP synthase. Modification occurs optimally at 37 degrees C and is inhibited by 5 mM 3',5'-ADP and 2 mM EDTA. An ACP construct (matACP) lacking the transit peptide was also converted to the holoACP form in an organelle-free assay, independent of precursor cleavage. The matACP construct was used to monitor the chromatographic separation of the holoACP synthase from the transit peptidase. Superose 12 gel filtration analysis indicates that the holoACP synthase has an apparent Mr of approximately 50,000. Using fractions enriched for the holoACP synthase it was demonstrated that the precursor of ACP is also modified in the presence of CoA and subsequently can be proteolytically processed directly to holoACP. Kinetic analysis, however, indicates that removal of the transit peptide is a much faster reaction than phosphopantetheine addition, suggesting that apoACP is the primary substrate for the chloroplast holoACP synthase in vivo.

The sequence of the entF gene which codes for the serine activating enzyme in enterobactin biosynthesis is reported. The gene encodes a protein with a calculated molecular weight of 142,006 and shares homologies with the small subunits of gramicidin S synthetase and tyrocidine synthetase. We have subcloned and overexpressed entF in a multicopy plasmid and attempted to demonstrate L-serine-dependent ATP-[32P]PPi exchange activity and its participation in enterobactin biosynthesis, but the overexpressed enzyme appears to be essentially inactive in crude extract. A partial purification of active EntF from wild-type Escherichia coli, however, has confirmed the expected activities of EntF. In a search for possible causes for the low level of activity of the overexpressed enzyme, we have discovered that EntF contains a covalently bound phosphopantetheine cofactor.

The C-terminal region of a multifunctional polypeptide from the 6-deoxyerythronolide B synthase of Saccharopolyspora erythraea is predicted to contain an acyl carrier protein and a thioesterase or acyltransferase activity [Cortes, J., Haydock, S. F., Roberts, G. A., Bevitt, D. J. & Leadlay, P. F. (1990) Nature 348, 176-178]. Site-directed mutagenesis by means of the polymerase chain reaction was used to construct an efficient pT7-based expression plasmid for this domain. The recently developed technique of electrospray mass spectrometry was used to demonstrate that the purified protein had not been post-translationally modified by attachment of a 4'-phosphopantetheine group. However, treatment with the serine proteinase inhibitor phenylmethylsulphonyl fluoride led to highly selective labelling of the predicted active site of the thioesterase or acyltransferase.

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 (≤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.

FDH (10-formyltetrahydrofolate dehydrogenase, Aldh1L1, EC 1.5.1.6) converts 10-formyltetrahydrofolate (10-formyl-THF) to tetrahydrofolate and CO(2) in a NADP(+)-dependent reaction. It is a tetramer of four identical 902 amino acid residue subunits. The protein subunit is a product of a natural fusion of three unrelated genes and consists of three distinct domains. The N-terminal domain of FDH (residues 1-310) carries the folate binding site and shares sequence homology and structural topology with other enzymes utilizing 10-formyl-THF as a substrate. In vitro it functions as 10-formyl-THF hydrolase, and evidence indicate that this activity is a part of the overall FDH mechanism. The C-terminal domain of FDH (residues 400-902) originated from an aldehyde dehydrogenase-related gene and is capable of oxidation of short-chain aldehydes to corresponding acids. Similar to classes 1 and 2 aldehyde dehydrogenases, this domain exists as a tetramer and defines the oligomeric structure of the full-length enzyme. The two catalytic domains are connected by an intermediate linker (residues 311-399), which is a structural and functional homolog of carrier proteins possessing a 4'-phosphopantetheine prosthetic group. In the FDH mechanism, the intermediate linker domain transfers a formyl, covalently attached to the sulfhydryl group of the phosphopantetheine arm, from the N-terminal domain to the C-terminal domain. The overall FDH mechanism is a coupling of two sequential reactions, a hydrolase and a formyl dehydrogenase, bridged by a substrate transfer step. In this mechanism, one domain provides the folate binding site and a hydrolase catalytic center to remove the formyl group from the folate substrate, another provides a transfer vehicle between catalytic centers and the third one contributes the dehydrogenase machinery further oxidizing formyl to CO(2).

The actinorhodin (act) synthase acyl carrier protein (ACP) from Streptomyces coelicolor plays a central role in polyketide biosynthesis. Polyketide intermediates are bound to the free sulfhydryl group of a phosphopantetheine arm that is covalently linked to a conserved serine residue in the holo form of the ACP. The solution NMR structures of both the apo and holo forms of the ACP are reported, which represents the first high resolution comparison of these two forms of an ACP. Ensembles of twenty apo and holo structures were calculated and yielded atomic root mean square deviations of well-ordered backbone atoms to the average coordinates of 0.37 and 0.42 A, respectively. Three restraints defining the protein to the phosphopantetheine interface were identified. Comparison of the apo and holo forms revealed previously undetected conformational changes. Helix III moved towards helix II (contraction of the ACP), and Leu43 on helix II subtly switched from being solvent exposed to forming intramolecular interactions with the newly added phosphopantetheine side chain. Tryptophan fluorescence and S. coelicolor fatty acid synthase (FAS) holo-synthase (ACPS) assays indicated that apo-ACP has a twofold higher affinity (K(d) of 1.1 muM) than holo-ACP (K(d) of 2.1 muM) for ACPS. Site-directed mutagenesis of Leu43 and Asp62 revealed that both mutations affect binding, but have differential affects on modification by ACPS. Leu43 mutations in particular strongly modulate binding affinity for ACPS. Comparison of apo- and holo-ACP structures with known models of the Bacillus subtilis FAS ACP-holo-acyl carrier protein synthase (ACPS) complex suggests that conformational modulation of helix II and III between apo- and holo-ACP could play a role in dissociation of the ACP-ACPS complex.

Coenzyme A (CoA) and its thioesters play a diverse array of roles in biologic systems. The enzymes that catalyze reactions of CoA are of interest for a variety of reasons, including, but not limited to, their potential as drug targets. The enzymology of CoA biosynthesis is now well understood, and the genes for all of the enzymes involved have been identified. Thioesters are inherently reactive toward acyl transfer reactions and toward reactions involving deprotonation of the α-carbon, and these are the primary reactivity patterns in enzyme-catalyzed reactions of CoA thioesters. CoA utilizing enzymes have been widely studied mechanistically and structurally. Analogs of the natural CoA thioester substrates have been widely used in these studies, including thioesters of unnatural or uncommon acyl groups as well as a large number of analogs in which the thioester is replaced with alternative functionality. Recent technical applications of CoA have included the tagging of carrier protein domains and carrier protein fusions with tagged phosphopantetheine derivatives transferred enzymatically from the corresponding tagged CoA derivatives using promiscuous phosphopantetheinyl transferases.

The acyl carrier protein (ACP) phosphodiesterase of Escherichia coli catalyzes the hydrolytic cleavage of the 4'-phosphopantetheine residue from ACP, with the generation of apo-ACP (P. R. Vagelos and A. R. Larrabee, J. Biol. Chem. 242:1776-1781, 1967). Although it has been postulated to play a role in the regulation of fatty acid synthesis, presently available evidence makes this unlikely, and its physiological function requires further investigation. We have now purified the enzyme from E. coli more than 3,000-fold and have identified it as a protein of Mr 25,000, as judged from its migration during electrophoresis in gels containing sodium dodecyl sulfate. The enzyme has remarkable thermostability, being protected against irreversible inactivation at 90 degrees C by the presence of sodium dodecyl sulfate. A partial sequence of the amino terminus of the enzyme is as follows: H2N-Ser-Lys-Val-Leu-Val-Leu-Lys-Ser-?-Ile-Leu-Ala-Gly-Tyr-Ser-. Other properties of the enzyme are also described.

Import of the acyl carrier protein (ACP) precursor into the chloroplast resulted in two products of about 14 kilodalton (kD) and 18 kD when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Time course experiments indicate that the latter is a modification derivative of the 14-kD peptide after the removal of the transit peptide. Substitution of serine 38 by alanine, eliminating the phosphopantetheine prosthetic group attachment site of ACP, produced a precursor mutant that gave rise to only the 14-kD peptide during import, showing that the modified form depends on the presence of serine 38. Furthermore, these results demonstrate that the prosthetic group is not essential for ACP translocation across the envelope or proteolytic processing. Analysis of the products of import by nondenaturing, conformationally sensitive gels showed reversal of the relative mobility of the 14-kD peptide and the modified form, raising the possibility that the modification is the addition of the phosphopantetheine. Proteolytic processing and the modification reaction were reconstituted in an organelle-free assay. The addition of coenzyme A to the organelle-free assay completely converted the 14-kD peptide to the modified form at 10 micromolar, and this only occurred with the wild-type substrate. Reciprocally, treatment of the products of a modification reaction with Escherichia coli phosphodiesterase converted the modified ACP from back to the 14-kD peptide. These results strongly support the conclusion that there is a holo-ACP synthase in the soluble compartment of the chloroplast capable of transferring the phosphopantetheine of coenzyme A to ACP.

Mass spectrometry (MS) is an important tool for studying non-ribosomal peptide, polyketide, and fatty acid biosynthesis. Here we describe a new approach using multi-stage tandem MS on a common ion trap instrument to obtain high-resolution measurements of the masses of substrates and intermediates bound to phosphopantetheinylated (holo) carrier proteins. In particular, we report the chemical formulas of 12 diagnostic MS(3) fragments of the phosphopantetheine moiety ejected from holo carrier proteins during MS(2). We demonstrate our method by observing the formation of holo-AcpC, a putative acyl carrier protein from Streptococcus agalactiae.

Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) are found in bacteria, fungi, and plants, in the main, producing antibiotics. They are macromolecular machines that rely on the activity of thioesterases to produce biologically active small molecules. They are of particular interest as an assembly system that might be adapted for the production of novel bioactive compounds with possible therapeutic activity. Frueh et al. have solved the structure of a carrier protein — part of the EntF NRPS subunit of enterobactin synthetase — bound to a type I thioesterase. (Type I thioesterases catalyse the final 'release' step of the small molecule from the NRPS or PKS machinery.) The structure reveals that part of the thioesterase can flip open to reveal the carrier-protein binding site of the enzyme; this movement allows the tether of the carrier protein to access the active site. Koglin et al. determined the structure of conformational sub-states of a thioesterase II enzyme. Type II thioesterases are required to regenerate a functional 4'-phosphopantetheine cofactor when it gets mis-primed by reacting with acetyl- and short chain acyl-residues. Comparison with the structures of type I thioesterases reveals the basis for substrate selectivity and the different modes of interaction of the two types of thioesterases with thiolation domains. The three-dimensional structure of a type II thioesterase, free and in complex with a thiolation domain, is determined. Comparison with type I thioesterases reveal the basis for substrate selectivity and the different modes of interaction of the two types of thioesterases with thiolation domains. Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) found in bacteria, fungi and plants use two different types of thioesterases for the production of highly active biological compounds1,2. Type I thioesterases (TEI) catalyse the release step from the assembly line3 of the final product where it is transported from one reaction centre to the next as a thioester linked to a 4′-phosphopantetheine (4′-PP) cofactor that is covalently attached to thiolation (T) domains4,5,6,7,8,9. The second enzyme involved in the synthesis of these secondary metabolites, the type II thioesterase (TEII), is a crucial repair enzyme for the regeneration of functional 4′-PP cofactors of holo-T domains of NRPS and PKS systems10,11,12. Mispriming of 4′-PP cofactors by acetyl- and short-chain acyl-residues interrupts the biosynthetic system. This repair reaction is very important, because roughly 80% of CoA, the precursor of the 4′-PP cofactor, is acetylated in bacteria13. Here we report the three-dimensional structure of a type II thioesterase from Bacillus subtilis free and in complex with a T domain. Comparison with structures of TEI enzymes3,14 shows the basis for substrate selectivity and the different modes of interaction of TEII and TEI enzymes with T domains. Furthermore, we show that the TEII enzyme exists in several conformations of which only one is selected on interaction with its native substrate, a modified holo-T domain.

The design, synthesis, and biochemical characterization of a mechanism-based aryl carrier protein (ArCP) affinity probe that selectively modifies the terminal thiol of the aryl carrier protein phosphopantetheine (Ppant) prosthetic group is described. Labeling of the aryl carrier protein was shown to require the cognate adenylating enzyme to channel the affinity probe onto the Ppant cofactor. The selective labeling was established by observation of the phosphopantetheinyl ejection ion via MS/MS, and the probe was also found to stabilize an interaction between an aryl carrier protein and adenylating enzyme by an electrophoretic mobility shift assay.

In the multifunctional fungal fatty acid synthase (FAS), the acyl carrier protein (ACP) domain shuttles reaction intermediates covalently attached to its prosthetic phosphopantetheine group between the different enzymatic centers of the reaction cycle. Here, we report the structure of the Saccharomyces cerevisiae FAS determined at 3.1 angstrom resolution with its ACP stalled at the active site of ketoacyl synthase. The ACP contacts the base of the reaction chamber through conserved, charge-complementary surfaces, which optimally position the ACP toward the catalytic cleft of ketoacyl synthase. The conformation of the prosthetic group suggests a switchblade mechanism for acyl chain delivery to the active site of the enzyme.

The metabolic cofactor coenzyme A (CoA) gained renewed attention because of its roles in neurodegeneration, protein acetylation, autophagy and signal transduction. The long-standing dogma is that eukaryotic cells obtain CoA exclusively via the uptake of extracellular precursors, especially vitamin B5, which is intracellularly converted through five conserved enzymatic reactions into CoA. This study demonstrates an alternative mechanism that allows cells and organisms to adjust intracellular CoA levels by using exogenous CoA. Here CoA was hydrolyzed extracellularly by ectonucleotide pyrophosphatases to 4'-phosphopantetheine, a biologically stable molecule able to translocate through membranes via passive diffusion. Inside the cell, 4'-phosphopantetheine was enzymatically converted back to CoA by the bifunctional enzyme CoA synthase. Phenotypes induced by intracellular CoA deprivation were reversed when exogenous CoA was provided. Our findings answer long-standing questions in fundamental cell biology and have major implications for the understanding of CoA-related diseases and therapies.

Nonribosomal peptide synthetases (NRPSs) are modular, multi-domain, enzymatic assembly lines that combine simple substrates to synthesize complex natural products (e.g. penicillin, bacitracin, yersiniabactin). Substrates are each covalently attached to so-called carrier proteins (CP) via a post-translationally introduced phosphopantetheine arm. CPs present the substrates to catalytic domains in a series of transient, sequential domain/domain and domain/substrate interactions during biosynthesis. Thus, understanding the mechanism of NRPS assembly line synthesis necessitates both characterizing fleeting molecular interactions and unraveling the molecular determinants for the directionality of these interactions. Unfortunately, the large molecular weight of NRPS domains, the presence of dynamics between domains and within domains, and the lability of tethered substrates have precluded studying these processes at the molecular level. Here, we present methods that permitted us to overcome these limitations and we discuss our findings. First, by exploiting the non-invasive nature of NMR we found that chemical substrates interact with their carrier proteins and these interactions may modulate domain communication to provide directionality. Second, we present a method that facilitates resonance assignments in large proteins. By applying covariance to conventional spectra, simplified correlation maps permit to identify sequential residues or to assign side-chain resonances with a simple visual inspection. Hence, resonances of 37 kDa and 53 kDa NRPS domains could be assigned unambiguously, enabling us to describe molecular signatures of transient interactions involving these domains.

Aldehyde oxidoreductase (carboxylic acid reductase (Car)) catalyzes the magnesium-, ATP-, and NADPH-dependent reduction of carboxylic acids to their corresponding aldehydes. Heterologous expression of the car gene in Escherichia coli afforded purified recombinant enzyme with a specific activity nearly 50-fold lower than that of purified native Nocardia sp. enzyme. The 5-fold increase in specific activity obtained by incubating purified recombinant Car with CoA and Nocardia cell-free extracts indicated that post-translational phosphopantetheinylation of Car is required for maximum enzyme activity. Nocardia phosphopantetheine transferase (PPTase) expressed in E. coli was isolated and characterized. When incubated with [(3)H]acetyl-CoA and Nocardia PPTase, the labeled acetylphosphopantetheine moiety was incorporated into recombinant Car. Coexpression of Nocardia Car and PPTase in E. coli gave a reductase with nearly 20-fold higher specific activity. Site-directed mutagenesis in which Ser(689) was replaced with Ala resulted in an inactive Car mutant. The results show that Car expressed in Escherichia coli is an apoenzyme that is converted to a holoenzyme by post-translational modification via phosphopantetheinylation. Doubly recombinant resting E. coli cells efficiently reduce vanillic acid to vanillin.

Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) regulates the daily rhythm in the production of melatonin and is therefore an attractive target for pharmacologic modulation of the synthesis of this hormone. Previously prepared bisubstrate analogs show potent inhibition of AANAT but have unfavorable pharmacokinetic properties due to the presence of phosphate groups which prevents transfer across the plasma membrane. Here, we examine a bis-pivaloyloxymethylene (POM)-tryptamine-phosphopantetheine prodrug (2) and its biotransformations in vitro by homogenates and pineal cells. Compound 2 is an efficient porcine liver esterase substrate for POM cleavage in vitro although cyclization of the phosphate moiety is a potential side product. Tryptamine phosphopantetheine (3) is converted to tryptamine-coenzyme A (CoA) bisubstrate analog (1) by human phosphoribosyl pyrophosphate amidotransferase (PPAT) and dephosphocoenzyme A kinase (DPCK) in vitro. Compound 2 was found to inhibit melatonin production in rat pineal cell culture. It was also found that the POM groups are readily removed to generate 3; however, further processing to tryptamine-CoA (1) is much slower in pineal extracts or cell culture. Implications for CoA prodrug development based on the strategy used here are discussed.

Acyl carrier protein (ACP) synthase (AcpS) catalyzes the transfer of the 4′-phosphopantetheine moiety from coenzyme A (CoA) onto a serine residue of apo-ACP, resulting in the conversion of apo-ACP to the functional holo-ACP. The holo form of bacterial ACP plays an essential role in mediating the transfer of acyl fatty acid intermediates during the biosynthesis of fatty acids and phospholipids. AcpS is therefore an attractive target for therapeutic intervention. In this study, we have purified and characterized the AcpS enzymes from Escherichia coli , Streptococcus pneumoniae , and Mycoplasma pneumoniae , which exemplify gram-negative, gram-positive, and atypical bacteria, respectively. Our gel filtration column chromatography and cross-linking studies demonstrate that the AcpS enzyme from M. pneumoniae , like E. coli enzyme, exhibits a homodimeric structure, but the enzyme from S. pneumoniae exhibits a trimeric structure. Our biochemical studies show that the AcpS enzymes from M. pneumoniae and S. pneumoniae can utilize both short- and long-chain acyl CoA derivatives but prefer long-chain CoA derivatives as substrates. On the other hand, the AcpS enzyme from E. coli can utilize short-chain CoA derivatives but not the long-chain CoA derivatives tested. Finally, our biochemical studies show that M. pneumoniae AcpS is kinetically a very sluggish enzyme compared with those from E. coli and S. pneumoniae . Together, the results of these studies show that the AcpS enzymes from different bacterial species exhibit different native structures and substrate specificities with regard to the utilization of CoA and its derivatives. These findings suggest that AcpS from different microorganisms plays a different role in cellular physiology.

Acyl Carrier Protein (ACP) from the malaria parasite, Plasmodium falciparum (PfACP) in its holo form is found to exist in two conformational states in solution. Unique 3D solution structures of holo-PfACP have been determined for both equilibrium conformations, using high-resolution NMR methods. Twenty high-resolution solution structures for each of the two forms of holo-PfACP have been determined on the basis of 1226 and 1218 unambiguously assigned NOEs (including NOEs between 4 '-phosphopantetheine prosthetic group (4 '-PP) and protein), 55 backbone dihedral angles and 26 hydrogen bonds. The atomic rmsd values of the determined structures of two equilibrium forms, about the mean coordinates of the backbone and heavy atoms, are 0.48 +/- 0.09 and 0.92 +/- 0.10 and 0.49 +/- 0.08 and 0.97 +/- 0.11 angstrom, respectively. The interaction of 4 '-PP with the polypeptide backbone is reported here for the first time for any of the ACPs. The structures of holo-PfACP consist of three well-defined helices that are tightly packed. The structured regions of the molecule are stabilized by extensive hydrophobic interactions. The difference between the two forms arises from a reorientation of the 4 '-PP group. The enthalpy difference between the two forms, although small, implies that a conformational switch is essential for the activation of holo-ACP. Sequence and structures of holo-PfACP have been compared with those of the ACPs from type I and type II fatty acid biosynthesis pathways (FAS), in particular with the ACP from rat and the butyryl-ACP from E. coli. The PfACP structure, thus determined has several novel features hitherto not seen in other ACPs.

Acyl carrier protein (ACP) is a cofactor in a variety of biosynthetic pathways, including fatty acid metabolism. Thus, it is of interest to determine structures of physiologically relevant ACP-fatty acid complexes. We report here the NMR solution structures of spinach ACP with decanoate (10:0-ACP) and stearate (18:0-ACP) attached to the 4'-phosphopantetheine prosthetic group. The protein in the fatty acid complexes adopts a single conformer, unlike apo- and holo-ACP, which interconvert in solution between two major conformers. The protein component of both 10:0- and 18:0-ACP adopts the four-helix bundle topology characteristic of ACP, and a fatty acid binding cavity was identified in both structures. Portions of the protein close in space to the fatty acid and the 4'-phosphopantetheine were identified using filtered/edited NOESY experiments. A docking protocol was used to generate protein structures containing bound fatty acid for 10:0- and 18:0-ACP. In both cases, the predominant structure contained fatty acid bound down the center of the helical bundle, in agreement with the location of the fatty acid binding pockets. These structures demonstrate the conformational flexibility of spinach ACP and suggest how the protein changes to accommodate its myriad binding partners.

Protein microarrays are an attractive approach for the high-throughput analysis of protein function, but their impact on proteomics has been limited by the technical difficulties associated with their generation. Here we demonstrate that fusion proteins of O6-alkylguanine-DNA alkyltransferase (AGT) can be used for the simple and reliable generation of protein microarrays for the analysis of protein function. Important features of the approach are the selectivity of the covalent immobilization; this allows for direct immobilization of proteins out of cell extracts, and the option both to label and to immobilize AGT fusion proteins, which allows for direct screening for protein-protein interactions between different AGT fusion proteins. In addition to the identification of protein-protein interactions, AGT-based protein microarrays can be used for the characterization of small molecule-protein interactions or post-translational modifications. The potential of the approach was demonstrated by investigating the post-translational modification of acyl carrier protein (ACP) from E. coli by different phosphopantetheine transferases (PPTases), yielding insights into the role of selected ACP amino acids in the ACP-PPTase interaction. © 2006 Wiley-VCH Verlag GmbH & Co. KGaA.

Pantothenic acid and biotin in vitamin supplements are obtained by chemical synthesis rather than by purification from natural sources. Isobutyraldehyde, formaldehyde and cyanide are used as starting materials in the chemical synthesis of pantothenic acid; the intermediate D-pantolactone is condensed with s-alanine to produce pantothenic acid. The majority of pantothenic acid in foodstuffs is present as CoA or 4'-phosphopantetheine; both these compounds are hydrolyzed by pyrophosphatase and phosphatase in the intestinal lumen to release pantetheine. Biotin in foods is largely protein bound; binding is mediated by an amide linkage between the valeric acid side chain in biotin and e-amino groups in lysines. Studies using jejunal segments from rats provided evidence that intestinal biotin uptake is mediated by both saturable and nonsaturable components. Calcium salts of D-pantothenate are the typical sources of pantothenic acid in oral vitamin supplements. Dexpanthenol is a synthetic analog of pantothenic acid that is commonly used for topical applications.

[structure: see text] D-Pantetheine and D-phosphopantetheine, precursors to coenzyme A, have been synthesized though a linear sequence from three modules (M1-M3) in 9 and 10 steps, respectively. These routes provide access to analogues of coenzyme A containing modified cystamines, beta-alanines, and pantoic acid residues. All three modules were joined using conventional methods of peptide synthesis. The chiral component, M3, was derived from D-pantolactone.

The specific and covalent labeling of fusion proteins with synthetic mols. opens up new ways to study protein function in the living cell. Here the authors present a novel method that allows for the specific and exclusive extracellular labeling of proteins on the surfaces of live cells with a large variety of synthetic mols. including fluorophores, protein ligands, or quantum dots. The approach is based on the specific labeling of fusion proteins of acyl carrier protein with synthetic mols. through post-translational modification catalyzed by phosphopantetheinetransferase. The specificity and versatility of the labeling should allow it to become an important tool for studying and manipulating cell surface proteins and for complementing existing approaches in cell surface engineering. [on SciFinder (R)]

4'-Phosphopantetheinyl transferases (PPTases) are essential for the production of fatty acids by fatty acid synthases (primary metabolism) and natural products by nonribosomal peptide synthetases and polyketide synthases (secondary metabolism). These systems contain carrier proteins (CPs) for the covalent binding of reaction intermediates during synthesis. PPTases transfer the 4'-phosphopantetheine moiety from coenzyme A (CoA) onto conserved serine residues of the apo-CPs to convert them to their functionally active holo form. In bacteria, two types of PPTases exist that are evolutionary related but differ in their substrate spectrum. Acyl carrier protein synthases (AcpSs) recognize CPs from primary metabolism, whereas Sfp- (surfactin production-) type PPTases have a preference for CPs of secondary metabolism. Previous investigations showed that a peptidyl carrier protein (PCP) of secondary metabolism can be altered to serve as substrate for AcpS. We demonstrate here that a single mutation in PCP suffices for the modification of this CP by AcpS, and we have identified by mutational analysis several other PCP residues and two AcpS residues involved in substrate discrimination by this PPTase. These altered PCPs were still capable of serving their designated function in NRPS modules, and selective use of AcpS or Sfp leads to production of two different products by a trimodular NRPS.

The activation of apo-peptidyl carrier proteins (PCPs) of nonribosomal peptide synthetases (NRPSs), apo-acyl carrier proteins (ACPs) of polyketide synthases (PKSs), and fatty acid synthases (FASs) to their active holo form is accomplished with dedicated 4'-phosphopantetheinyl transferases (PPTases). They catalyze the transfer of the essential prosthetic group 4'-phosphopantetheine (4'-Ppant) from coenzyme A (CoA) to a highly conserved serine residue in all PCPs and ACPs. PPTases, based on sequence and substrate specifity, have been classified into three types: bacterial holo-acyl carrier protein synthase (AcpS), fatty acid synthase of eukaryotes (FAS2) and Sfp, a PPTase of secondary metabolism. The recently solved crystal structures of AcpS and Sfp-type PPTases with CoA revealed a common alpha + beta-fold with a beta(1)alpha(3)beta(2) motif and similarities in CoA binding and polymerization mode. However, it was not possible to discern neither the PCP binding region of Sfp nor the priming reaction mechanism from the Sfp-CoA cocrystal. In this work, we provide a model for the reaction mechanism based on mutational analysis of Sfp that suggests a reaction mechanism in which the highly conserved E151 deprotonates the hydroxyl group of the invariant serine of PCP. That, in turn, acts as a nucleophile to attack the beta-phosphate of CoA. The Sfp mutants K112, E117, and K120 further revealed that the loop region between beta4 and alpha5 (residues T111-S124) in Sfp is the PCP binding region. Also, residues T44, K75, S89, H90, D107, E109, E151, and K155 that have been shown in the Sfp-CoA cocrystal structure to coordinate CoA are now all confirmed by mutational and biochemical analysis.

Here we report the first biochemical characterization of a recombinant nonribosomal peptide cyclase of a streptomycete, the model actinomycete Streptomyces coelicolor A3(2). This bacterium produces the calcium-dependent antibiotic (CDA), which is a branched cyclic macrolactone belonging to the group of acidic lipopeptides. The recombinant CDA3 cyclase from CDA synthetase efficiently catalyzes ring formation of linear peptidyl thioester substrates based on a sequence analogous to natural CDA. Four leaving groups were attached to the C-terminus of the undecapeptide: coenzyme A (CoA), phosphopantetheine, N-acetylcysteamine (SNAC), and thiophenol. The best rates for cyclization were determined for the thiophenol substrate, revealing that chemical reactivity is more important than cofactor recognition. The cyclase catalyzes the formation of two regioisomeric macrolactones, which arise from simultaneous nucleophilic attack of the two adjacent Thr(2) and Ser(1) residues onto the C-terminus of the acyl-enzyme intermediate. This relaxed regioselectivity has not been observed for any other recombinant NRPS or PKS cyclases so far. Substitution of either Ser(1) or Thr(2) by alanine led to selective formation of a decapeptide or undecapeptide lactone ring. In contrast to that, CDA3 cyclase strictly retains stereoselectivity for both nucleophiles, accepting only l-configured Ser(1) and Thr(2) for cyclization. Further, our studies provide evidence for the crucial role of N-terminal fatty acyl groups of lipopeptides in controlling the regio- and chemoselectivity of enzyme-catalyzed macrocyclization. Elongation of the fatty acyl group of our thioester substrate from C(2) to C(6) as in CDA turned the relaxed regioselectivity into a strict regioselectivity, yielding solely the decapeptide lactone ring with a significantly improved cyclization-to-hydrolysis ratio.

Pantothenate (vitamin B5) is the precursor for the biosynthesis of the phosphopantetheine moiety of coenzyme A and acyl carrier protein, and is synthesised in Escherichia coli by four enzymic reactions. Ketopantoate hydroxymethyltransferase (KPHMT) and pantothenate synthetase (PtS) catalyse the first and last steps, respectively. Two genes encoding KPHMT and one for PtS were identified in the Arabidopsis thaliana genome, and cDNAs for all three genes were amplified by PCR. The cDNAs were able to complement their respective E. coli auxotrophs, demonstrating that they encoded functional enzymes. Subcellular localisation of the proteins was investigated using green fluorescent protein (GFP) fusions and confocal microscopy. The two KPHMT-GFP fusion proteins were targeted exclusively to mitochondria, whereas PtS-GFP was found in the cytosol. This implies that there must be transporters for pathway intermediates. KPHMT enzyme activity could be measured in purified mitochondria from both pea leaves and Arabidopsis suspension cultures. We investigated whether Arabidopsis encoded homologues of the remaining two pantothenate biosynthesis enzymes from E. coli, l-aspartate-alpha-decarboxylase (ADC) and ketopantoate reductase (KPR). No homologue of ADC could be identified using either conventional blast or searches with the program fugue in which the structure of the E. coli ADC was compared to all the annotated proteins in Arabidopsis. ADC also appears to be absent from the genome of the yeast, Saccharomyces cerevisiae, by the same criteria. In contrast, a putative Arabidopsis oxidoreductase with some similarity to KPR was identified with fugue.

Selective incorporation of extender units in modular polyketide synthases is primarily controlled by acyl transferase (AT) domains. The AT domains catalyze transacylation of the extender unit from acyl-CoA to the phosphopantetheine arm of an acyl carrier protein (ACP) domain in the same module. New methods that can modulate the extender unit specificity of individual modules with minimal structural or kinetic perturbations in the engineered module are desirable for the efficient biosynthesis of novel natural product analogues. We have demonstrated that transacylation of malonyl groups onto an AT-null form of a mutant modular polyketide synthase by malonyl-CoA:ACP transacylase is an effective strategy for the engineered biosynthesis of site specifically modified polyketides. Using this strategy, 6-deoxyerythronolide B synthase was engineered to exclusively produce 2-desmethyl-6-deoxyerythronolide B. The productivity of the modified system was comparable to that of the wild-type synthase in vitro and in vivo.

Non-ribosomal peptide synthetases (NRPS) are modular assembly lines catalysing the synthesis of many small peptides in microbes. Genetic replacements of domains or modules in NRPS encoded by gene clusters in Bacillus sp. with corresponding domains or modules from foreign NRPS have led in several cases to the in vivo synthesis of peptides with predicted amino acid substitutions. Fusion points were in variable regions between C- and A- or between T- and C-domains. Successful insertions of whole modules using fusion points in conserved regions internal to functional domains have also been reported. For studying the role of C-, A-, T- and TE (thioesterase)-domains in NRPS, several bi- and trimodular model-NRPS derived from natural NRPS systems were constructed and obtained after expression in E. coli with coexpression of a 4'- phosphopantetheine transferase or in suitable hosts such as the Streptomyces. Such enzymes were shown to catalyse in vitro synthesis of di- and tripeptides, respectively, with and without turnover depending on the presence of Te-domains. The enzymatic analysis revealed the mechanisms of the domains and proved their functional autonomy suggesting the possibility to use any NRPS interdomain region for fusions. Nevertheless, recombinant synthesis of longer and more complex peptides will still be restricted to alteration of existing structures by manipulations of NRPS gene clusters located on chromosomes or artificial chromosomes. Besides targeted replacements of domains and modules, reprogramming of NRPS by altering the substrate specificities of A-domains is a promising tool for the future to get novel peptides.

Here we report a new experimental approach to characterize recombinant nonribosomal peptide cyclases that do not show activity with conventional N-acetylcysteamine (SNAC) substrates. To explore the great potential of these domains for the catalysis of cell-free cyclization reactions, the new strategy takes advantage of the direct interaction between the natural substrate where the peptide chain is attached to the phosphopantetheine arm of the peptidyl carrier protein (PCP) and the peptide cyclase. A prerequisite for this reaction is the promiscuity of the Bacillus subtilis phosphopantetheinyl transferase Sfp for loading chemically synthesized peptidyl-coenzyme A substrates instead of the smaller natural substrate coenzyme A (CoASH) onto apoPCP. With this novel method we were able to characterize the regioselectivity of branched-chain cyclization catalyzed by the fengycin cyclase, which displays no activity with peptidyl-SNAC substrates.

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.

Phosphopantetheine adenylyltransferase (PPAT) regulates the key penultimate step in the essential coenzyme A (CoA) biosynthetic pathway. PPAT catalyzes the reversible transfer of an adenylyl group from Mg 2+ :ATP to 4′-phosphopantetheine to form 3′-dephospho-CoA (dPCoA) and pyrophosphate. The high-resolution crystal structure of PPAT complexed with CoA has been determined. Remarkably, CoA and the product dPCoA bind to the active site in distinct ways. Although the phosphate moiety within the phosphopantetheine arm overlaps, the pantetheine arm binds to the same pocket in two distinct conformations, and the adenylyl moieties of these two ligands have distinct binding sites. Moreover, the PPAT:CoA crystal structure confirms the asymmetry of binding to the two trimers within the hexameric enzyme. Specifically, the pantetheine arm of CoA bound to one protomer within the asymmetric unit displays the dPCoA-like conformation with the adenylyl moiety disordered, whereas CoA binds the twofold-related protomer in an ordered and unique fashion.

Nonribosomal peptide synthetases (NRPSs) assemble structurally complex peptides from simple building blocks such as amino and carboxyl acids. Product release by macrocyclization or hydrolysis is catalyzed by a thioesterase domain that is an integrated part of the NRPS enzyme. A second thioesterase of type II (TEII) encoded by a distinct gene associated with the NRPS cluster was previously shown by means of gene disruption to be important for efficient product formation. However, the actual role of TEIIs in nonribosomal peptide synthesis remained obscure. Here we report the biochemical characterization of two such TEII enzymes that are associated with the synthetases of the peptide antibiotics surfactin (TEII srf ) and bacitracin (TEII bac ). Both enzymes were shown to efficiently regenerate misacylated thiol groups of 4′-phosphopantetheine (4′PP) cofactors attached to the peptidyl carrier proteins (PCPs) of NRPSs. For TEII srf , a K M of 0.9 μM and a k cat of 95 min −1 was determined for acetyl-PCP hydrolysis. Both enzymes could also hydrolyze aminoacyl or peptidyl PCPs, intermediates of nonribosomal peptide synthesis. However, this reaction is unlikely to be of physiological relevance. Similar intermediates of the primary metabolism such as CoA derivatives and acetyl-acyl carrier proteins of fatty acid synthesis were also not significantly hydrolyzed, as investigated with TEII srf . These findings support a model in which the physiological role of TEIIs in nonribosomal peptide synthesis is the regeneration of misacylated NRPS, which result from the apo to holo conversion of NRPS enzymes because of the promiscuity of dedicated 4′PP transferases that use not only free CoA, but also acyl-CoAs as 4′PP donors.

Phosphopantetheinyl-dependent carrier proteins are part of fatty-acid synthases (primary metabolism), polyketide synthases, and non-ribosomal peptide synthetases (secondary metabolism). For these proteins to become functionally active, they need to be primed with the 4′-phosphopantetheine moiety of coenzyme A by a dedicated phosphopantetheine transferase (PPTase). Most organisms that employ more than one phosphopantetheinyl-dependent pathway also have more than one PPTase. Typically, one of these PPTases is optimized for the modification of carrier proteins of primary metabolism and rejects those of secondary metabolism (AcpS-type PPTases), whereas the other, Sfp-type PPTase, efficiently modifies carrier proteins involved in secondary metabolism. We present here a new type of PPTase, the carrier protein synthase of Pseudomonas aeruginosa, an organism that harbors merely one PPTase, namely PcpS. Gene deletion experiments clearly show that PcpS is essential for growth of P. aeruginosa, and biochemical data indicate its association with both fatty acid synthesis and siderophore metabolism. At first sight, PcpS is a PPTase of the monomeric Sfp-type and was consequently expected to have catalytic properties typical for this type of enzyme. However, in vitro characterization of PcpS with natural protein partners and non-cognate substrates revealed that its catalytic properties differ significantly from those of Sfp. Thus, the situation in P. aeruginosa is not simply the result of the loss of an AcpS-type PPTase. PcpS exhibits high catalytic efficiency with the carrier protein of fatty acid synthesis and shows a reduced although significant conversion rate of the carrier proteins of non-ribosomal peptide synthetases from their apo to holo form. This association with enzymes of primary and secondary metabolism indicates that PcpS belongs to a new sub-class of PPTases.

Phosphopantetheine-dependent enzymes are a group of multifunctional or individual enzymes including fatty acid synthases, polyketide synthases and nonribosomal polypeptide synthases, all of which utilize the 4′-phosphopantetheine moiety attached to an acyl carrier protein or a peptide carrier protein. The terminal thiol group of 4′-phosphopantetheine is covalently attached to any of a number of acyl groups in the form of a thiol ester to facilitate acyl transfer and nucleophilic activation. Keywords: 4′-phosphopentetheine; coenzyme A (CoA); thiol ester; C–C bond formation; acyl carrier protein; fatty acid synthase; nonribosomal polypeptide synthase; polypeptide carrier protein; antibiotic biosynthesis

Acyl carrier proteins carry the lipid substrate to the enzymes of the fatty acid synthase system. Crystals of Escherichia coli acyl carrier protein to which a butyryl group has been attached via a thioester link to the phosphopantetheine prosthetic arm have been obtained by the hanging-drop vapour-diffusion method. These crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 27.6, b = 41.6, c = 63.7 A. The asymmetric unit appears to contain one subunit, corresponding to a packing density of 2.1 A(3) Da(-1). Crystals of the selenomethionine-substituted (SeMet) protein were obtained using different conditions and belong to space group P6(3), with unit-cell parameters a = b = 63.4, c = 37.0 A, alpha = beta = 90, gamma = 120 degrees and with a monomer in the asymmetric unit (V(M) = 2.5 A(3) Da(-1)). Crystals of a SeMet butyryl-ACP I62M variant were obtained using the conditions for the native protein. Like the native crystals, these belong to space group P2(1)2(1)2(1) and have unit-cell parameters a = 27.3, b = 41.9, c = 64.5 A. A data set suitable for MAD phasing was collected from the crystals of the I62M variant to 1.8 A resolution on the ESRF beamline ID14-4.

Page 11242. In refs 2–5, the cited journal name should be Nature, not Nat. Methods. The corrected full citations are presented here [the list is renumbered as (2−5) in this erratum]

Acyl carrier protein (ACP) plays an essential role inside bacterial multicomponent enzyme systems. Like a little delivery truck, it transports substrates, intermediates, and products among catalytic domains as the enzyme systems biosynthesize natural products. And it uses a phosphopantetheine (Ppant) group as a working “arm” to actually carry out these tasks. Researchers have now devised a technique that monitors Ppant motions occurring in nanoseconds or picoseconds—orders of magnitude faster than could be detected previously. The study might lead to a better molecular understanding of the workings of ACP-containing enzyme systems such as polyketide synthases, nonribosomal peptide synthases, and fatty acid synthases. That understanding could also make it easier to engineer multienzyme systems to produce customized natural-product-like compounds for drug discovery. While ACP makes its rounds, the Ppant arm keeps changing shape to interact with individual enzyme units and help protect reactive intermediates...

Phosphopantetheinyl transferases (PPTases) are essential enzymes that catalyze covalent attachment of the 4'-phosphopantetheine (4'-PP) moiety from coenzyme A (CoA) to a conserved serine residue on acyl (ACP) and peptidyl carrier proteins (PCP) [1]. This post-translational modification converts the inactive apo-carrier proteins to the functional form, shuttling the intermediates of biosynthetic reactions catalyzed by fatty acid synthases (FAS), polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPSs). In Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), two PPTases, AcpS (type-I) and PptT (type-II), are involved in the biosynthesis of essential lipids, virulence factors and siderophores, activating over 20 target proteins [2]. These two proteins have been shown to be independently essential, suggesting that PPTases could be targeted against tuberculosis [2, 3]. We have expressed the Mtb-PptT protein as a fusion protein with maltose binding protein (MBP). The use of the MBP-PptT fusion protein overcame stability and solubility problems and resulted in successful crystallization. The structure of Mtb-PptT in complex with CoA was determined from the crystal of the fusion protein, solved at 1.75 Å resolution. Excellent electron density is present for all parts of the CoA molecule, revealing a conserved CoA-binding mode. Conformational and charge distribution differences in the putative ACP binding cleft, however, suggest a different mode of ACP binding compared to other homologues. This is the first and only three-dimensional structure of a type-II PPTase from pathogenic bacteria, providing structural features that can be exploited in drug development when compared with its human counterpart.

Fatty acids and polyketides are synthesized by mechanistically and evolutionarily related multienzyme systems. Their carbon chain backbones are synthesized via repeated decarboxylative condensations of alpha-carboxylated building blocks onto a growing acyl chain. These alpha-carboxylated building blocks are transferred from the corresponding coenzyme A thioesters onto the phosphopantetheine arm of an acyl carrier protein (ACP) by acyl transferases, which operate by a ping-pong mechanism involving an acyl-O-serine intermediate. In the course of our studies on the malonyl-CoA:ACP transacylase (MAT) from Streptomyces coelicolor, we observed that an active-site Ser (97) --> Ala mutant retains activity as well as the ability to be covalently labeled by (14)C malonyl-CoA. Here we demonstrate that an alternative, catalytically competent nucleophile exists in the active site of this enzyme. Next to the active-site serine is a histidine residue that is conserved in some, but not all acyl transferases. The H96A mutant is also active and can be labeled, but an H96A/S97A double mutant is inactive and cannot be labeled. The ability of H96 to form a malonyl-imidazole adduct was confirmed by proteolysis, followed by radio-HPLC and mass spectrometric analysis of the S97A mutant enzyme. Kinetic analysis revealed that the k(cat) of the S97A mutant was within 10-fold that of the wild-type enzyme, whereas the K(M)s of the two enzymes were comparable. Sequence comparison with the E. coli MAT (whose X-ray structure is known) led to the identification of H201 as the putative base in the serine-histidine catalytic dyad of the S. coelicolor enzyme. The absence of MAT activity in the H201A mutant and the detection of weak activity in the H201Q mutant was consistent with this proposal. The implications of this unexpected finding are discussed.

Bacillus subtilis was reported to produce the catecholic siderophore itoic acid (2,3-dihydroxybenzoate (DHB)-glycine) in response to iron deprivation. However, by inspecting the DNA sequences of the genes dhbE, dhbB, and dhbF as annotated by the B. subtilis genome project to encode the synthetase complex for the siderophore assembly, various sequence errors within the dhbF gene were predicted and confirmed by re-sequencing. According to the corrected sequence, dhbF encodes a dimodular instead of a monomodular nonribosomal peptide synthetase. We have heterologously expressed, purified, and assayed the substrate selectivity of the recombinant proteins DhbB, DhbE, and DhbF. DhbE, a stand-alone adenylation domain of 59.9 kDa, activates, in an ATP-dependent reaction, DHB, which is subsequently transferred to the free thiol group of the cofactor phosphopantetheine of the bifunctional isochorismate lyase/aryl carrier protein DhbB. The third synthetase, DhbF, is a dimodular nonribosomal peptide synthetase of 264 kDa that specifically adenylates threonine and, to a lesser extent, glycine and that covalently loads both amino acids onto their corresponding peptidyl carrier domains. To functionally link the dhb gene cluster to siderophore synthesis, we have disrupted the dhbF gene. Comparative mass spectrometric analysis of culture extracts from both the wild type and the dhbF mutant led to the identification of a mass peak at m/z 881 ([M-H](1-)) that corresponds to a cyclic trimeric ester of DHB-glycine-threonine.