RESUMEN
The delineation of the complex biosynthesis of the potent antibiotic mupirocin, which consists of a mixture of pseudomonic acids (PAs) isolated from Pseudomonas fluorescens NCIMB 10586, presents significant challenges and the timing and mechanisms of several key transformations remain elusive. Particularly intriguing are the steps that process the linear backbone from the initial polyketide assembly phase to generate the first cyclic intermediate PA-B. These include epoxidation as well as incorporation of the tetrahydropyran (THP) ring and fatty acid sidechain required for biological activity. Here, we show that the mini-module MmpE performs a rare online (ACP-substrate) epoxidation and is integrated ('in-cis') into the polyketide synthase via a docking domain. A linear polyketide fragment with 6 asymmetric centres was synthesised using a convergent approach and used to demonstrate substrate flux via an atypical KS0 and a previously unannotated ACP (MmpE_ACP). MmpE_ACP-bound synthetic substrates were critical in demonstrating successful epoxidation in vitro by the purified MmpE oxidoreductase domain. Alongside feeding studies, these results confirm the timing as well as chain length dependence of this selective epoxidation. These mechanistic studies pinpoint the location and nature of the polyketide substrate prior to the key formation of the THP ring and esterification that generate PA-B.
RESUMEN
The presence of ß-branches in the structure of polyketides that possess potent biological activity underpins the widespread importance of this structural feature. Kalimantacin is a polyketide antibiotic with selective activity against staphylococci, and its biosynthesis involves the unprecedented incorporation of three different and sequential ß-branching modifications. We use purified single and multi-domain enzyme components of the kalimantacin biosynthetic machinery to address inâ vitro how the pattern of ß-branching in kalimantacin is controlled. Robust discrimination of enzyme products required the development of a generalisable assay that takes advantage of 13 Câ NMR of a single 13 C label incorporated into key biosynthetic mimics combined with favourable dynamic properties of an acyl carrier protein. We report a previously unassigned modular enoyl-CoA hydratase (mECH) domain and the assembly of enzyme constructs and cascades that are able to generate each specific ß-branch.
Asunto(s)
Radioisótopos de Carbono/análisis , Enoil-CoA Hidratasa/química , Enoil-CoA Hidratasa/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Carbamatos/química , Carbamatos/metabolismo , Ácidos Grasos Insaturados/química , Ácidos Grasos Insaturados/metabolismo , Modelos Moleculares , Especificidad por SustratoRESUMEN
Polyketides are a large class of bioactive natural products with a wide range of structures and functions. Polyketides are biosynthesized by large, multidomain enzyme complexes termed polyketide synthases (PKSs). One of the primary challenges when studying PKSs is the high reactivity of their poly-ß-ketone substrates. This has hampered structural and mechanistic characterization of PKS-polyketide complexes, and, as a result, little is known about how PKSs position the unstable substrates for proper catalysis while displaying high levels of regio- and stereospecificity. As a first step toward a general plan to use oxetanes as carbonyl isosteres to broadly interrogate PKS chemistry, we describe the development and application of an oxetane-based PKS substrate mimic. This enabled the first structural determination of the acyl-enzyme intermediate of a ketosynthase (KS) in complex with an inert extender unit mimic. The crystal structure, in combination with molecular dynamics simulations, led to a proposed mechanism for the unique activity of DpsC, the priming ketosynthase for daunorubicin biosynthesis. The successful application of an oxetane-based polyketide mimic suggests that this novel class of probes could have wide-ranging applications to the greater biosynthetic community interested in the mechanistic enzymology of iterative PKSs.
Asunto(s)
Éteres Cíclicos/química , Sondas Moleculares/química , Sintasas Poliquetidas/química , Policétidos/química , Sitios de Unión , Éteres Cíclicos/metabolismo , Sondas Moleculares/metabolismo , Estructura Molecular , Sintasas Poliquetidas/metabolismo , Policétidos/metabolismo , Especificidad por SustratoRESUMEN
Three-coordinate bipyridyl complexes of gold, [(κ2-bipy)Au(η2-C2H4)][NTf2], are readily accessed by direct reaction of 2,2'-bipyridine (bipy), or its derivatives, with the homoleptic gold ethylene complex [Au(C2H4)3][NTf2]. The cheap and readily available bipyridyl ligands facilitate oxidative addition of aryl iodides to the Au(I) center to give [(κ2-bipy)Au(Ar)I][NTf2], which undergo first aryl-zinc transmetalation and second C-C reductive elimination to produce biaryl products. The products of each distinct step have been characterized. Computational techniques are used to probe the mechanism of the oxidative addition step, offering insight into both the origin of the reversibility of this process and the observation that electron-rich aryl iodides add faster than electron-poor substrates. Thus, for the first time, all steps that are characteristic of a conventional intermolecular Pd(0)-catalyzed biaryl synthesis are demonstrated from a common monometallic Au complex and in the absence of directing groups.
RESUMEN
Pseudomonas mesoacidophila ATCC 31433 is a Gram-negative bacterium, first isolated from Japanese soil samples, that produces the monobactam isosulfazecin and the ß-lactam-potentiating bulgecins. To characterize the biosynthetic potential of P. mesoacidophila ATCC 31433, its complete genome was determined using single-molecule real-time DNA sequence analysis. The 7.8-Mb genome comprised four replicons, three chromosomal (each encoding rRNA) and one plasmid. Phylogenetic analysis demonstrated that P. mesoacidophila ATCC 31433 was misclassified at the time of its deposition and is a member of the Burkholderia cepacia complex, most closely related to Burkholderia ubonensis The sequenced genome shows considerable additional biosynthetic potential; known gene clusters for malleilactone, ornibactin, isosulfazecin, alkylhydroxyquinoline, and pyrrolnitrin biosynthesis and several uncharacterized biosynthetic gene clusters for polyketides, nonribosomal peptides, and other metabolites were identified. Furthermore, P. mesoacidophila ATCC 31433 harbors many genes associated with environmental resilience and antibiotic resistance and was resistant to a range of antibiotics and metal ions. In summary, this bioactive strain should be designated B. cepacia complex strain ATCC 31433, pending further detailed taxonomic characterization.IMPORTANCE This work reports the complete genome sequence of Pseudomonas mesoacidophila ATCC 31433, a known producer of bioactive compounds. Large numbers of both known and novel biosynthetic gene clusters were identified, indicating that P. mesoacidophila ATCC 31433 is an untapped resource for discovery of novel bioactive compounds. Phylogenetic analysis demonstrated that P. mesoacidophila ATCC 31433 is in fact a member of the Burkholderia cepacia complex, most closely related to the species Burkholderia ubonensis Further investigation of the classification and biosynthetic potential of P. mesoacidophila ATCC 31433 is warranted.
Asunto(s)
Complejo Burkholderia cepacia/genética , Pseudomonas/genética , Antibacterianos/farmacología , Complejo Burkholderia cepacia/clasificación , Complejo Burkholderia cepacia/efectos de los fármacos , ADN Bacteriano/genética , Farmacorresistencia Bacteriana , Regulación Bacteriana de la Expresión Génica/fisiología , Genoma Bacteriano/genética , Filogenia , Pseudomonas/clasificación , Pseudomonas/efectos de los fármacosRESUMEN
Type I polyketide synthases often use programmed ß-branching, via enzymes of a 'hydroxymethylglutaryl-CoA synthase (HCS) cassette', to incorporate various side chains at the second carbon from the terminal carboxylic acid of growing polyketide backbones. We identified a strong sequence motif in acyl carrier proteins (ACPs) where ß-branching is known to occur. Substituting ACPs confirmed a correlation of ACP type with ß-branching specificity. Although these ACPs often occur in tandem, NMR analysis of tandem ß-branching ACPs indicated no ACP-ACP synergistic effects and revealed that the conserved sequence motif forms an internal core rather than an exposed patch. Modeling and mutagenesis identified ACP helix III as a probable anchor point of the ACP-HCS complex whose position is determined by the core. Mutating the core affects ACP functionality, whereas ACP-HCS interface substitutions modulate system specificity. Our method for predicting ß-carbon branching expands the potential for engineering new polyketides and lays a basis for determining specificity rules.
Asunto(s)
Proteína Transportadora de Acilo/química , Proteína Transportadora de Acilo/metabolismo , Secuencia Conservada , Hidroximetilglutaril-CoA Sintasa/metabolismo , Policétidos/metabolismo , Proteína Transportadora de Acilo/genética , Secuencias de Aminoácidos , Modelos Moleculares , Conformación Molecular , Policétidos/químicaRESUMEN
Atropisomerism of pharmaceutical compounds is a challenging area for drug discovery programs (Angew. Chem., Int. Ed. 2009, 48, 6398-6401). Strategies for dealing with these compounds include raising the energy barrier to atropisomerization in order to develop the drug as a single isomer (Tetrahedron 2004, 60, 4337-4347) or reducing the barrier to rotation and developing a mixture of rapidly interconverting isomers (Chirality 1996, 8, 364-371). Commonly, however, the atropisomers will be differentiated in terms of their affinity for a given protein target, and it is therefore important to rapidly identify the most active component prior to further compound development. We present equilibrium dialysis and saturation transfer difference NMR (STD-NMR) as techniques for assessing relative affinities of an atropisomeric mixture against antiapoptotic protein targets Bcl-2 and Bcl-xL. These techniques require no prior separation of the mixture of compounds and are therefore rapid and simple approaches. We also explore the use of noncovalent mass spectrometry for determining KD values of individual atropisomers separated from the equilibrium mixture and compare the results to solution-phase measurements. Results from equilibrium dialysis, STD-NMR, and noncovalent mass spectrometry are all in excellent agreement and provide complementary information on differential binding, amplification of the strongest binders, and KD values.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Espectrometría de Masas/métodos , Preparaciones Farmacéuticas/análisis , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Proteína bcl-X/análisis , Preparaciones Farmacéuticas/metabolismo , Unión Proteica/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína bcl-X/metabolismoRESUMEN
Common to all FASs, PKSs and NRPSs is a remarkable component, the acyl or peptidyl carrier protein (A/PCP). These take the form of small individual proteins in type II systems or discrete folded domains in the multi-domain type I systems and are characterized by a fold consisting of three major α-helices and between 60-100 amino acids. This protein is central to these biosynthetic systems and it must bind and transport a wide variety of functionalized ligands as well as mediate numerous protein-protein interactions, all of which contribute to efficient enzyme turnover. This review covers the structural and biochemical characterization of carrier proteins, as well as assessing their interactions with different ligands, and other synthase components. Finally, their role as an emerging tool in biotechnology is discussed.
Asunto(s)
Proteína Transportadora de Acilo/química , Proteína Transportadora de Acilo/metabolismo , Sintasas Poliquetidas/química , Sintasas Poliquetidas/metabolismo , Humanos , Datos de Secuencia Molecular , Estructura Molecular , Policétidos/química , Policétidos/metabolismo , Conformación ProteicaRESUMEN
Most mesenchymal neoplasms of the gastrointestinal tract are currently classified as gastrointestinal stromal tumors (GIST). Gastrointestinal stromal tumors are diagnosed by immunopositivity for CD117, CD34, and DOG1.1, with or without molecular analyses. According to the World Health Organization classification, the diagnosis of primary leiomyosarcomas of the gastrointestinal tract is so rare that there are no significant data on demographic, clinical, or gross features of this tumor. A comprehensive literature search was performed to identify gastrointestinal leiomyosarcomas. Searches were limited to the past 12 years because definitive tools to differentiate leiomyosarcomas from GIST were introduced in the late 1990s. Cases were included only if convincing data were presented. Six cases of esophageal leiomyosarcoma and 5 cases of gastric leiomyosarcoma were confirmed. Furthermore, 26 cases of leiomyosarcoma of the small bowel, 11 cases of the colon, and 8 cases arising in the rectum were identified. Finally, 28 cases of infantile and adolescent leiomyosarcoma were reviewed. Although survival analysis is precluded by small case numbers and limited survival data availability, the trend identifies that increased size and mitotic activity portends to a worse prognosis in small bowel leiomyosarcomas. Colonic leiomyosarcomas appear to be aggressive tumors, regardless of tumor size and mitotic activity. Rectal leiomyosarcomas present as smaller tumors with favorable prognosis. Leiomyosarcomas in post-GIST era are rare tumors of the gastrointestinal tract with distinctive clinicopathologic characteristics. Owing to different treatment options, it is necessary to accurately differentiate these from GIST, using a combination of histologic appearance, presence of smooth muscle antigens, and absence of specific GIST immunomarkers.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias Gastrointestinales/patología , Tumores del Estroma Gastrointestinal/patología , Leiomiosarcoma/patología , Adulto , Biomarcadores de Tumor/genética , ADN de Neoplasias/química , ADN de Neoplasias/genética , Diagnóstico Diferencial , Gastrectomía , Neoplasias Gastrointestinales/genética , Neoplasias Gastrointestinales/metabolismo , Neoplasias Gastrointestinales/cirugía , Tumores del Estroma Gastrointestinal/genética , Tumores del Estroma Gastrointestinal/metabolismo , Tracto Gastrointestinal/patología , Humanos , Leiomiosarcoma/genética , Leiomiosarcoma/metabolismo , Leiomiosarcoma/cirugía , Perdida de Seguimiento , Masculino , Índice Mitótico , Clasificación del Tumor , Pronóstico , Análisis de Secuencia de ADN , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Neoplasias Gástricas/cirugía , Células del Estroma/patologíaRESUMEN
We report a case demonstrating a rare finding associated with a relatively common injury. Lipomata are rarely found within tendon sheaths; but in the case of our patient, at the time of operative repair for a ruptured Achilles tendon, we found a fatty growth within the tendon sheath. The diagnosis of a lipoma was confirmed by histology. Although uncommon, it remains important to be aware of the existence of neoplastic growths within tendon sheaths and to establish the exact nature of these growths by histological analysis.
Asunto(s)
Tendón Calcáneo/patología , Lipoma/diagnóstico , Enfermedades Musculares/diagnóstico , Tendón Calcáneo/lesiones , Tendón Calcáneo/cirugía , Estudios de Seguimiento , Humanos , Lipoma/complicaciones , Lipoma/cirugía , Masculino , Persona de Mediana Edad , Enfermedades Musculares/complicaciones , Enfermedades Musculares/cirugía , Procedimientos Ortopédicos/métodos , Prótesis e Implantes , Rotura/complicaciones , Traumatismos de los Tendones/complicaciones , Traumatismos de los Tendones/patología , Traumatismos de los Tendones/cirugíaRESUMEN
In type II polyketide synthases (PKSs), which typically biosynthesize several antibiotic and antitumor compounds, the substrate is a growing polyketide chain, shuttled between individual PKS enzymes, while covalently tethered to an acyl carrier protein (ACP): this requires the ACP interacting with a series of different enzymes in succession. During biosynthesis of the antibiotic actinorhodin, produced by Streptomyces coelicolor, one such key binding event is between an ACP carrying a 16-carbon octaketide chain (actACP) and a ketoreductase (actKR). Once the octaketide is bound inside actKR, it is likely cyclized between C7 and C12 and regioselective reduction of the ketone at C9 occurs: how these elegant chemical and conformational changes are controlled is not yet known. Here, we perform protein-protein docking, protein NMR, and extensive molecular dynamics simulations to reveal a probable mode of association between actACP and actKR; we obtain and analyze a detailed model of the C7-C12-cyclized octaketide within the actKR active site; and we confirm this model through multiscale (QM/MM) reaction simulations of the key ketoreduction step. Molecular dynamics simulations show that the most thermodynamically stable cyclized octaketide isomer (7R,12R) also gives rise to the most reaction competent conformations for ketoreduction. Subsequent reaction simulations show that ketoreduction is stereoselective as well as regioselective, resulting in an S-alcohol. Our simulations further indicate several conserved residues that may be involved in selectivity of C7-12 cyclization and C9 ketoreduction. Detailed insights obtained on ACP-based substrate presentation in type II PKSs can help design ACP-ketoreductase systems with altered regio- or stereoselectivity.
RESUMEN
The transfer of the phosphopantetheine chain from coenzyme A (CoA) to the acyl carrier protein (ACP), a key protein in both fatty acid and polyketide synthesis, is catalyzed by ACP synthase (AcpS). Streptomyces coelicolor AcpS is a doubly promiscuous enzyme capable of activation of ACPs from both fatty acid and polyketide synthesis and catalyzes the transfer of modified CoA substrates. Five crystal structures have been determined, including those of ligand-free AcpS, complexes with CoA and acetyl-CoA, and two of the active site mutants, His110Ala and Asp111Ala. All five structures are trimeric and provide further insight into the mechanism of catalysis, revealing the first detailed structure of a group I active site with the essential magnesium in place. Modeling of ACP binding supported by mutational analysis suggests an explanation for the promiscuity in terms of both ACP partner and modified CoA substrates.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Modelos Moleculares , Streptomyces coelicolor/enzimología , Transferasas (Grupos de Otros Fosfatos Sustitutos)/química , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Dominio Catalítico/genética , Cristalografía por Rayos X , Proteínas de Escherichia coli/química , Ligandos , Magnesio/química , Datos de Secuencia Molecular , Unión Proteica/genética , Pliegue de Proteína , Streptomyces coelicolor/genética , Especificidad por Sustrato/genética , Transferasas/química , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genéticaRESUMEN
Transcription of the 74 kb Pseudomonas fluorescens mupirocin [pseudomonic acid (PA)] biosynthesis cluster depends on quorum sensing-dependent regulation via the LuxI/LuxR homologues MupI/MupR. To facilitate analysis of novel PAs from pathway mutants, we investigated factors that affect mup gene expression. First, the signal produced by MupI was identified as N-(3-oxodecanoyl)homoserine lactone, but exogenous addition of this molecule did not activate mupirocin production prematurely nor did expression of mupI in trans increase metabolite production. Second, we confirmed that mupX, encoding an amidase/hydrolase that can degrade N-acylhomoserine lactones, is also required for efficient expression, consistent with its occurrence in a regulatory module linked to unrelated genes in P. fluorescens. Third, and most significantly, mupR expression in trans to wild type and mutants can increase production of antibiotic and novel intermediates up to 17-fold.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Mupirocina/metabolismo , Pseudomonas fluorescens/fisiología , Percepción de Quorum , Regulación hacia Arriba , Antibacterianos/biosíntesis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Pseudomonas fluorescens/genéticaRESUMEN
Acyl (peptidyl) carrier protein (ACP or PCP) is a crucial component involved in the transfer of thiol ester-bound intermediates during the biosynthesis of primary and secondary metabolites such as fatty acids, polyketides, and nonribosomal peptides. Although many carrier protein three-dimensional structures have been determined, to date there is no model available for a fungal type I polyketide synthase ACP. Here we report the solution structure of the norsolorinic acid synthase (NSAS) holo ACP domain that has been excised from the full-length multifunctional enzyme. NSAS ACP shows similarities in three-dimensional structure with other type I and type II ACPs, consisting of a four-helix bundle with helices I, II, and IV arranged in parallel. The N-terminus of helix III, however, is unusually hydrophobic, and Phe1768 and Leu1770 pack well with the core of the protein. The result is that unlike other carrier proteins, helix III lies almost perpendicular to the three major helices. Helix III is well-defined by numerous NMR-derived distance restraints and may be less flexible than counterparts in type II FAS and PKS ACPs. When the holo ACP is derivatized with a hexanoyl group, only minor changes are observed between the HSQC spectra of the two ACP species and no NOEs are observed for this hydrophobic acyl group. Along with the mammalian type I FAS, this further strengthens the view that type I ACPs do not show any significant affinity for hydrophobic (nonpolar) chain assembly intermediates attached via the 4'-phosphopantetheine prosthetic group.
Asunto(s)
Proteína Transportadora de Acilo/química , Sintasas Poliquetidas/química , Streptomyces coelicolor/enzimología , Acilación , Secuencia de Aminoácidos , Aporfinas/metabolismo , Holoenzimas/química , Holoenzimas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Sintasas Poliquetidas/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , SolucionesRESUMEN
Acyl carrier proteins are vital for the biosynthesis of fatty acids and polyketides. The mupirocin biosynthetic cluster of Pseudomonas fluorescens encodes eleven type I ACPs embedded in its multifunctional polyketide synthase (PKS) proteins plus five predicted type II ACPs (mAcpA-E) that are known to be essential for mupirocin biosynthesis by deletion and complementation analysis. MupN is a putative Sfp-type phosphopantetheinyl transferase. Overexpression of three type I and three type II mupirocin ACPs in Escherichia coli, with or without mupN, followed by mass spectroscopy revealed that MupN can modify both mupirocin type I and type II ACPs to their holo-form. The endogenous phosphopantetheinyl transferase of E. coli modified mAcpA but not mAcpC or D. Overexpression of the type II ACPs in macp deletion mutants of the mupirocin producer P. fluorescens 10586 showed that they cannot substitute for each other while hybrids between mAcpA and mAcpB indicated that, at least for mAcpB, the C-terminal domain determines functional specificity. Amino acid alignments identified mACPs A and D as having C-terminal extensions. Mutation of these regions generated defective ACPs, the activity of which could be restored by overexpression of the macp genes on separate plasmids.
Asunto(s)
Proteína Transportadora de Acilo/metabolismo , Antibacterianos/biosíntesis , Proteínas Bacterianas/metabolismo , Mupirocina/biosíntesis , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Proteína Transportadora de Acilo/genética , Secuencia de Aminoácidos , Antibacterianos/química , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Ácidos Grasos/biosíntesis , Macrólidos/metabolismo , Datos de Secuencia Molecular , Familia de Multigenes , Mupirocina/química , Mupirocina/farmacología , Mutación , Sintasas Poliquetidas/genética , Sintasas Poliquetidas/metabolismo , Dominios y Motivos de Interacción de Proteínas , Pseudomonas fluorescens/enzimología , Pseudomonas fluorescens/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genéticaRESUMEN
The insulin-like growth factor II/mannose-6-phosphate receptor (IGF2R) mediates trafficking of mannose-6-phosphate (M6P)-containing proteins and the mitogenic hormone IGF2. IGF2R also plays an important role as a tumor suppressor, as mutation is frequently associated with human carcinogenesis. IGF2 binds to domain 11, one of 15 extracellular domains on IGF2R. The crystal structure of domain 11 and the solution structure of IGF2 have been reported, but, to date, there has been limited success when using crystallography to study the interaction of IGFs with their binding partners. As an approach to investigate the interaction between IGF2 and IGF2R, we have used heteronuclear NMR in combination with existing mutagenesis data to derive models of the domain 11-IGF2 complex by using the program HADDOCK. The models reveal that the molecular interaction is driven by critical hydrophobic residues on IGF2 and IGF2R, while a ring of flexible, charged residues on IGF2R may modulate binding.
Asunto(s)
Proteínas/metabolismo , Receptor IGF Tipo 2/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Cartilla de ADN , Humanos , Factor II del Crecimiento Similar a la Insulina , Datos de Secuencia Molecular , Mutagénesis , Resonancia Magnética Nuclear Biomolecular , Reacción en Cadena de la Polimerasa , Unión Proteica , Conformación Proteica , Proteínas/química , Proteínas/genética , Homología de Secuencia de Aminoácido , Resonancia por Plasmón de SuperficieRESUMEN
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.