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We have identified an acyl-carrier protein, Rv0100, that is up-regulated in a dormancy model. This protein plays a critical role in the fatty acid biosynthesis pathway, which is important for energy storage and cell wall synthesis in Mycobacterium tuberculosis (MTB). Knocking out the Rv0100 gene resulted in a significant reduction of growth compared to wild-type MTB in the Wayne model of non-replicating persistence. We have also shown that Rv0100 is essential for the growth and survival of this pathogen during infection in mice and a macrophage model. Furthermore, knocking out Rv0100 disrupted the synthesis of phthiocerol dimycocerosates, the virulence-enhancing lipids produced by MTB and Mycobacterium bovis. We hypothesize that this essential gene contributes to MTB virulence in the state of latent infection. Therefore, inhibitors targeting this gene could prove to be potent antibacterial agents against this pathogen., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Gutka et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The loops of modular polyketide synthases (PKSs) serve diverse functions but are largely uncharacterized. They frequently contain amino acid repeats resulting from genetic events such as slipped-strand mispairing. Determining the tolerance of loops to amino acid changes would aid in understanding and engineering these multidomain molecule factories. Here, tandem repeats in the DNA encoding 949 modules within 129 cis-acyltransferase PKSs were cataloged, and the locations of the corresponding amino acids within the module were identified. The most frequently inserted interdomain loop corresponds with the updated module boundary immediately downstream of the ketosynthase (KS), while the loops bordering the dehydratase are nearly intolerant to such insertions. From the 949 modules, no repetitive sequence loop insertions are located within ACP, and only 2 reside within KS, indicating the sensitivity of these domains to alteration., (© 2021 Wiley Periodicals LLC.)

Aurantinins (ARTs) are antibacterial polyketides featuring a unique 6/7/8/5-fused tetracyclic ring system and a triene side chain with a carboxyl terminus. Here we identify the art gene cluster and dissect ART's C-methyl incorporation patterns to study its biosynthesis. During this process, an apparently redundant methyltransferase Art28 was characterized as a malonyl-acyl carrier protein O-methyltransferase, which represents an unusual on-line methyl esterification initiation strategy for polyketide biosynthesis. The methyl ester bond introduced by Art28 is kept until the last step of ART biosynthesis, in which it is hydrolyzed by Art9 to convert inactive ART 9B to active ART B. The cryptic reactions catalyzed by Art28 and Art9 represent a protecting group biosynthetic logic to render the ART carboxyl terminus inert to unwanted side reactions and to protect producing organisms from toxic ART intermediates. Further analyses revealed a wide distribution of this initiation strategy for polyketide biosynthesis in various bacteria., (© 2021. The Author(s).)

Background: Aeromonas hydrophila is a gram-negative bacterium and the major causative agent of the fish disease motile aeromonad septicemia (MAS). It uses N-acyl-homoserine lactone (AHL) quorum sensing signals to coordinate biofilm formation, motility, and virulence gene expression. The AHL signaling pathway is therefore considered to be a therapeutic target against pathogenic A. hydrophila infection. In A. hydrophila, AHL autoinducers biosynthesis are specifically catalyzed by an ACP-dependent AHL synthase AhyI using the precursors SAM and acyl-ACP. Our previously reported AhyI was heterologously expressed in E. coli, which showed the production characteristics of medium-long chain AHLs. This contradicted the prevailing understanding that AhyI was only a short-chain C 4 /C 6 -HSL synthase., Results: In this study, six linear acyl-ACP proteins with C-terminal his-tags were synthesized in Vibrio harveyi AasS using fatty acids and E. coli produced active holo-ACP proteins, and in vitro biosynthetic assays of six AHL molecules and kinetic studies of recombinant AhyI with a panel of four linear acyl-ACPs were performed. UPLC-MS/MS analyses indicated that AhyI can synthesize short-, medium- and long-chain AHLs from SAM and corresponding linear acyl-ACP substrates. Kinetic parameters measured using a DCPIP colorimetric assay, showed that there was a notable decrease in catalytic efficiency with acyl-chain lengths above C6, and hyperbolic or sigmoidal responses in rate curves were observed for varying acyl-donor substrates. Primary sequence alignment of the six representative AHL synthases offers insights into the structural basis for their specific acyl substrate preference. To further understand the acyl chain length preference of AhyI for linear acyl-ACP, we performed a structural comparison of three ACP-dependent LuxI homologs (TofI, BmaI1 and AhyI) and identified three key hydrophobic residues (I67, F125 and L157) which confer AhyI to selectively recognize native C 4 /C 6 -ACP substrates. These predictions were further supported by a computational Ala mutation assay., Conclusions: In this study, we have redefined AhyI as a multiple short- to long-chain AHL synthase which uses C 4 /C 6 -ACP as native acyl substrates and longer acyl-ACPs (C8 ~ C14) as non-native ones. We also theorized that the key residues in AhyI would likely drive acyl-ACP selective recognition.

Mycobacterium tuberculosis causes tuberculosis, a disease that kills over 1 million people each year. Its cell envelope is a common antibiotic target and has a unique structure due, in part, to two lipidated polysaccharides-arabinogalactan and lipoarabinomannan. Arabinofuranosyltransferase D (AftD) is an essential enzyme involved in assembling these glycolipids. We present the 2.9-Å resolution structure of M. abscessus AftD, determined by single-particle cryo-electron microscopy. AftD has a conserved GT-C glycosyltransferase fold and three carbohydrate-binding modules. Glycan array analysis shows that AftD binds complex arabinose glycans. Additionally, AftD is non-covalently complexed with an acyl carrier protein (ACP). 3.4- and 3.5-Å structures of a mutant with impaired ACP binding reveal a conformational change, suggesting that ACP may regulate AftD function. Mutagenesis experiments using a conditional knockout constructed in M. smegmatis confirm the essentiality of the putative active site and the ACP binding for AftD function., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Thermotoga maritima , a deep-branching hyperthermophilic bacterium, expresses an extraordinarily stable Thermotoga maritima acyl carrier protein ( Tm -ACP) that functions as a carrier in the fatty acid synthesis system at near-boiling aqueous environments. Here, to understand the hyperthermal adaptation of Tm -ACP, we investigated the structure and dynamics of Tm -ACP by nuclear magnetic resonance (NMR) spectroscopy. The melting temperature of Tm -ACP (101.4 °C) far exceeds that of other ACPs, owing to extensive ionic interactions and tight hydrophobic packing. The D59 residue, which replaces Pro/Ser of other ACPs, mediates ionic clustering between helices III and IV. This creates a wide pocket entrance to facilitate the accommodation of long acyl chains required for hyperthermal adaptation of the T. maritima cell membrane. Tm -ACP is revealed to be the first ACP that harbor an amide proton hyperprotected against hydrogen/deuterium exchange for I15. The hydrophobic interactions mediated by I15 appear to be the key driving forces of the global folding process of Tm -ACP. Our findings provide insights into the structural basis of the hyperthermal adaptation of ACP, which might have allowed T. maritima to survive in hot ancient oceans.

Salmonella is a facultative intracellular pathogen that invades epithelial cells of the intestine using the SPI-1 Type 3 secretion System (T3SS). Insertion of the SPI-1 T3SS translocon is facilitated by acylation of the translocator SipB, which involves a protein-protein interaction with the acyl carrier protein IacP. Using nuclear magnetic resonance and biological tests, we identified the residues of IacP that are involved in the interaction with SipB. Our results suggest that the 4'-phosphopantetheine group that functionalizes IacP participates in the interaction. Its solvent exposition may rely on two residues highly conserved in acyl carrier proteins associated with T3SS. This study is the first to address the specificity of acyl carrier proteins associated with T3SS., (© 2019 Federation of European Biochemical Societies.)

Acyl carrier proteins (ACPs) are essential to the production of fatty acids. In some species of marine bacteria, ACPs are arranged into tandem repeats joined by peptide linkers, an arrangement that results in high fatty acid yields. By contrast, Escherichia coli, a relatively low producer of fatty acids, uses a single-domain ACP. In this work, we have engineered the native E. coli ACP into tandem di- and tri-domain constructs joined by a naturally occurring peptide linker from the PUFA synthase of Photobacterium profundum. The size of these tandem fused ACPs was determined by size exclusion chromatography to be higher (21 kDa, 36 kDa and 141 kDa) than expected based on the amino acid sequence (12 kDa, 24 kDa and 37 kDa, respectively) suggesting the formation of a flexible extended conformation. Structural studies using small-angle X-ray scattering (SAXS), confirmed this conformational flexibility. The thermal stability for the di- and tri-domain constructs was similar to that of the unfused ACP, indicating a lack of interaction between domains. Lastly, E. coli cultures harboring tandem ACPs produced up to 1.6 times more fatty acids than wild-type ACP, demonstrating the viability of ACP fusion as a method to enhance fatty acid yield in bacteria.

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

Acyl carrier proteins (ACPs) play essential roles in the synthesis of fatty acids and transfer of long fatty acyl chains into complex lipids. The Enterococcus faecalis genome contains two annotated acp genes, called acpA and acpB AcpA is encoded within the fatty acid synthesis (fab) operon and appears essential. In contrast, AcpB is an atypical ACP, having only 30% residue identity with AcpA, and is not essential. Deletion of acpB has no effect on E. faecalis growth or de novo fatty acid synthesis in media lacking fatty acids. However, unlike the wild-type strain, where growth with oleic acid resulted in almost complete blockage of de novo fatty acid synthesis, the ΔacpB strain largely continued de novo fatty acid synthesis under these conditions. Blockage in the wild-type strain is due to repression of fab operon transcription, leading to levels of fatty acid synthetic proteins (including AcpA) that are insufficient to support de novo synthesis. Transcription of the fab operon is regulated by FabT, a repressor protein that binds DNA only when it is bound to an acyl-ACP ligand. Since AcpA is encoded in the fab operon, its synthesis is blocked when the operon is repressed and acpA thus cannot provide a stable supply of ACP for synthesis of the acyl-ACP ligand required for DNA binding by FabT. In contrast to AcpA, acpB transcription is unaffected by growth with exogenous fatty acids and thus provides a stable supply of ACP for conversion to the acyl-ACP ligand required for repression by FabT. Indeed, ΔacpB and ΔfabT strains have essentially the same de novo fatty acid synthesis phenotype in oleic acid-grown cultures, which argues that neither strain can form the FabT-acyl-ACP repression complex. Finally, acylated derivatives of both AcpB and AcpA were substrates for the E. faecalis enoyl-ACP reductases and for E. faecalis PlsX (acyl-ACP; phosphate acyltransferase). IMPORTANCE AcpB homologs are encoded by many, but not all, lactic acid bacteria ( Lactobacillales ), including many members of the human microbiome. The mechanisms regulating fatty acid synthesis by exogenous fatty acids play a key role in resistance of these bacteria to those antimicrobials targeted at fatty acid synthesis enzymes. Defective regulation can increase resistance to such inhibitors and also reduce pathogenesis., (Copyright © 2019 Zhu et al.)

The biosynthesis of the pimelate moiety of biotin in Escherichia coli requires two specialized proteins, BioC and BioH. However, the enzymes that have BioC- or BioH-like activities show remarkable sequence diversity among biotin-producing bacteria. Here, we report that the intracellular rickettsial pathogen Ehrlichia chaffeensis encodes two novel proteins, BioT and BioU, which functionally replace the E. coli BioC and BioH proteins, respectively. The desthiobiotin assays demonstrated that these two proteins make pimeloyl-acyl carrier protein (ACP) from the substrate malonyl-ACP with the aid of the FAS II pathway, through the expected pimeloyl-ACP methyl ester intermediate. BioT and BioU homologues seem restricted to the species of Ehrlichia and its close relative, Anaplasma. Taken together, the synthesis of the biotin precursor in E. chaffeensis appears to be catalyzed by two novel BioC- and BioH-like proteins.

Trans-acting acyltransferases (trans-ATs) are standalone enzymes that select and deliver extender units to polyketide synthase assembly lines. Accordingly, there is interest in leveraging trans-ATs as tools to regioselectively diversify polyketide structures. Yet, little is known regarding the extender unit and acyl carrier protein (ACP) specificity of trans-ATs, particularly those that utilize unusual ACP-linked extender units. For example, the biosynthesis of the antibiotic zwittermicin involves the trans-AT ZmaF, which is responsible for installing a rare ACP-linked aminomalonyl extender unit. Here, we developed a method to access a panel of non-natural and non-native ACP-linked extender units and used it to probe the promiscuity of ZmaF, revealing one of the most promiscuous ATs characterized to date. Furthermore, we demonstrated that ZmaF is highly orthogonal with respect to its ACP specificity, and the ability of ZmaF to trans-complement noncognate PKS modules was also explored. Together, these results set the stage for further engineering ZmaF as a tool for polyketide diversification.

The precursors of the diffusible signal factor (DSF) family signals of Xanthomonas campestris pv. campestris are 3-hydroxyacyl-acyl carrier protein (3-hydroxyacyl-ACP) thioesters having acyl chains of 12 to 13 carbon atoms produced by the fatty acid biosynthetic pathway. We report a novel 3-oxoacyl-ACP reductase encoded by the X. campestris pv. campestris XCC0416 gene ( fabG2 ), which is unable to participate in the initial steps of fatty acyl synthesis. This was shown by the failure of FabG2 expression to allow growth at the nonpermissive temperature of an Escherichia coli fabG temperature-sensitive strain. However, when transformed into the E. coli strain together with a plasmid bearing the Vibrio harveyi acyl-ACP synthetase gene ( aasS ), growth proceeded, but only when the medium contained octanoic acid. In vitro assays showed that FabG2 catalyzes the reduction of long-chain (≥C 8 ) 3-oxoacyl-ACPs to 3-hydroxyacyl-ACPs but is only weakly active with shorter-chain (C 4 , C 6 ) substrates. FabG1, the housekeeping 3-oxoacyl-ACP reductase encoded within the fatty acid synthesis gene cluster, could be deleted in a strain that overexpressed fabG2 but only in octanoic acid-supplemented media. Growth of the X. campestris pv. campestris Δ fabG1 strain overexpressing fabG2 required fabH for growth with octanoic acid, indicating that octanoyl coenzyme A is elongated by X. campestris pv. campestris fabH Deletion of fabG2 reduced DSF family signal production, whereas overproduction of either FabG1 or FabG2 in the Δ fabG2 strain restored DSF family signal levels. IMPORTANCE Quorum sensing mediated by DSF signaling molecules regulates pathogenesis in several different phytopathogenic bacteria, including Xanthomonas campestris pv. campestris DSF signaling also plays a key role in infection by the human pathogen Burkholderia cepacia The acyl chains of the DSF molecules are diverted and remodeled from a key intermediate of the fatty acid synthesis pathway. We report a Xanthomonas campestris pv. campestris fatty acid synthesis enzyme, FabG2, of novel specificity that seems tailored to provide DSF signaling molecule precursors., (Copyright © 2018 Hu et al.)

The marine bacterium Pseudoalteromonas tunicata produces the bipyrrole antibiotic tambjamine YP1. This natural product is built from common amino acid and fatty acid building blocks in a biosynthetic pathway that is encoded in the tam operon which contains 19 genes. The exact role that each of these Tam proteins plays in tambjamine biosynthesis is not known. Here, we provide evidence that TamA initiates the synthesis and controls the chain length of the essential tambjamine fatty amine tail. Sequence analysis suggests the unusual TamA is comprised of an N-terminal adenylation (ANL) domain fused to a C-terminal acyl carrier protein (ACP). Mass spectrometry analysis of recombinant TamA revealed the surprising presence of bound C11 and C12 acyl-adenylate intermediates. Acylation of the ACP domain was observed upon attachment of the phosphopantetheine (4'-PP) arm to the ACP. We also show that TamA can transfer fatty acids ranging in chain length from C6-C13 to an isolated ACP domain. Thus TamA bridges the gap between primary and secondary metabolism by linking fatty acid and pyrrole biosynthetic pathways.

Fatty acid biosynthesis (FAS) is a vital process in cells. Fatty acids are essential for cell assembly and cellular metabolism. Abnormal FAS directly correlates with cell growth delay and human diseases, such as metabolic syndromes and various cancers. The FAS system utilizes an acyl carrier protein (ACP) as a transporter to stabilize and shuttle the growing fatty acid chain throughout enzymatic modules for stepwise catalysis. Studying the interactions between enzymatic modules and ACP is, therefore, critical for understanding the biological function of the FAS system. However, the information remains unclear due to the high flexibility of ACP and its weak interaction with enzymatic modules. We present here a 2.55 Å crystal structure of type II FAS dehydratase FabZ in complex with holo-ACP, which exhibits a highly symmetrical FabZ hexamer-ACP 3 stoichiometry with each ACP binding to a FabZ dimer subunit. Further structural analysis, together with biophysical and computational results, reveals a novel dynamic seesaw-like ACP binding and catalysis mechanism for the dehydratase module in the FAS system, which is regulated by a critical gatekeeper residue (Tyr100 in FabZ) that manipulates the movements of the β-sheet layer. These findings improve the general understanding of the dehydration process in the FAS system and will potentially facilitate drug and therapeutic design for diseases associated with abnormalities in FAS.

Phosphopantetheinyl transferases (PPTases) play essential roles in both primary metabolisms and secondary metabolisms via post-translational modification of acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs). In this study, an industrial FK506 producing strain Streptomyces tsukubaensis L19, together with Streptomyces avermitilis, was identified to contain the highest number (five) of discrete PPTases known among any species thus far examined. Characterization of the five PPTases in S. tsukubaensis L19 unveiled that stw ACP, an ACP in a type II PKS, was phosphopantetheinylated by three PPTases FKPPT1, FKPPT3, and FKACPS; sts FAS ACP, the ACP in fatty acid synthase (FAS), was phosphopantetheinylated by three PPTases FKPPT2, FKPPT3, and FKACPS; TcsA-ACP, an ACP involved in FK506 biosynthesis, was phosphopantetheinylated by two PPTases FKPPT3 and FKACPS; FkbP-PCP, an PCP involved in FK506 biosynthesis, was phosphopantetheinylated by all of these five PPTases FKPPT1-4 and FKACPS. Our results here indicate that the functions of these PPTases complement each other for ACPs/PCPs substrates, suggesting a complicate phosphopantetheinylation network in S. tsukubaensis L19. Engineering of these PPTases in S. tsukubaensis L19 resulted in a mutant strain that can improve FK506 production.

Pamamycins are macrodiolides of polyketide origin with antibacterial activities. Their biosynthesis has been proposed to utilize succinate as a building block. However, the mechanism of succinate incorporation into a polyketide was unclear. Here, we report identification of a pamamycin biosynthesis gene cluster by aligning genomes of two pamamycin-producing strains. This unique cluster contains polyketide synthase (PKS) genes encoding seven discrete ketosynthase (KS) enzymes and one acyl-carrier protein (ACP)-encoding gene. A cosmid containing the entire set of genes required for pamamycin biosynthesis was successfully expressed in a heterologous host. Genetic and biochemical studies allowed complete delineation of pamamycin biosynthesis. The pathway proceeds through 3-oxoadipyl-CoA, a key intermediate in the primary metabolism of the degradation of aromatic compounds. 3-Oxoadipyl-CoA could be used as an extender unit in polyketide assembly to facilitate the incorporation of succinate., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Comparative genome analysis revealed seven uncharacterized genes, sven0909 to sven0915, adjacent to the previously identified chloramphenicol biosynthetic gene cluster (sven0916-sven0928) of Streptomyces venezuelae strain ATCC 10712 that was absent in a closely related Streptomyces strain that does not produce chloramphenicol. Transcriptional analysis suggested that three of these genes might be involved in chloramphenicol production, a prediction confirmed by the construction of deletion mutants. These three genes encode a cluster-associated transcriptional activator (Sven0913), a phosphopantetheinyl transferase (Sven0914), and a Na(+)/H(+) antiporter (Sven0915). Bioinformatic analysis also revealed the presence of a previously undetected gene, sven0925, embedded within the chloramphenicol biosynthetic gene cluster that appears to encode an acyl carrier protein, bringing the number of new genes likely to be involved in chloramphenicol production to four. Microarray experiments and synteny comparisons also suggest that sven0929 is part of the biosynthetic gene cluster. This has allowed us to propose an updated and revised version of the chloramphenicol biosynthetic pathway., (Copyright © 2014 Fernández-Martínez et al.)

Fluvirucin B1 , produced by Actinomadura vulgaris, is a 14-membered macrolactam active against a variety of infectious fungi as well as influenza A. Despite considerable interest from the synthetic community, very little information is available regarding the biosynthetic origins of the fluvirucins. Herein, we report the identification and initial characterization of the fluvirucin B1 polyketide synthase and related enzymes. The cluster consists of five extender modules flanked by an N-terminal acyl carrier protein and C-terminal thioesterase domain. All but one of the synthase modules contain the full complement of tailoring domains (ketoreductase, dehydratase, and enoyl reductase) as determined by sequence homology with known polyketide synthases. Acitve site analyses of several key components of the cluster are performed to further verify that this gene cluster is associated with production of fluvirucin B1 . This work will both open doors toward a better understanding of macrolactam formation and provide an avenue to genetics-based diversification of fluvirucin structure.

Unlabelled: Enoyl-acyl carrier protein (enoyl-ACP) reductase catalyzes the last step of the elongation cycle in the synthesis of bacterial fatty acids. The Enterococcus faecalis genome contains two genes annotated as enoyl-ACP reductases, a FabI-type enoyl-ACP reductase and a FabK-type enoyl-ACP reductase. We report that expression of either of the two proteins restores growth of an Escherichia coli fabI temperature-sensitive mutant strain under nonpermissive conditions. In vitro assays demonstrated that both proteins support fatty acid synthesis and are active with substrates of all fatty acid chain lengths. Although expression of E. faecalis fabK confers to E. coli high levels of resistance to the antimicrobial triclosan, deletion of fabK from the E. faecalis genome showed that FabK does not play a detectable role in the inherent triclosan resistance of E. faecalis. Indeed, FabK seems to play only a minor role in modulating fatty acid composition. Strains carrying a deletion of fabK grow normally without fatty acid supplementation, whereas fabI deletion mutants make only traces of fatty acids and are unsaturated fatty acid auxotrophs., Importance: The finding that exogenous fatty acids support growth of E. faecalis strains defective in fatty acid synthesis indicates that inhibitors of fatty acid synthesis are ineffective in countering E. faecalis infections because host serum fatty acids support growth of the bacterium.

Incubation of recombinant module 2 of the polyether nanchangmycin synthase (NANS), carrying an appended thioesterase domain, with the ACP-bound substrate (2RS)-2-methyl-3-ketobutyryl-NANS_ACP1 (2-ACP1) and methylmalonyl-CoA in the presence of NADPH gave diastereomerically pure (2S,4R)-2,4-dimethyl-5-ketohexanoic acid (4a). These results contrast with the previously reported weak discrimination by NANS module 2+TE between the enantiomers of the corresponding N-acetylcysteamine-conjugated substrate analogue (±)-2-methyl-3-ketobutyryl-SNAC (2-SNAC), which resulted in formation of a 5:3 mixture of 4a and its (2S,4S)-diastereomer 4b. Incubation of NANS module 2+TE with 2-ACP1 in the absence of NADPH gave unreduced 3,5,6-trimethyl-4-hydroxypyrone (3) with a k(cat) of 4.4 ± 0.9 min⁻¹ and a k(cat)/K(m) of 67 min⁻¹ mM⁻¹, corresponding to a ∼2300-fold increase compared to the k(cat)/K(m) for the diffusive substrate 2-SNAC. Covalent tethering of the 2-methyl-3-ketobutyryl thioester substrate to the NANS ACP1 domain derived from the natural upstream PKS module of the nanchangmycin synthase significantly enhanced both the stereospecificity and the kinetic efficiency of the sequential polyketide chain translocation and condensation reactions catalyzed by the ketosynthase domain of NANS module 2.

The life cycle of Dictyostelium discoideum is proposed to be regulated by expression of small metabolites. Genome sequencing studies have revealed a remarkable array of genes homologous to polyketide synthases (PKSs) that are known to synthesize secondary metabolites in bacteria and fungi. A crucial step in functional activation of PKSs involves their post-translational modification catalyzed by phosphopantetheinyl transferases (PPTases). PPTases have been recently characterized from several bacteria; however, their relevance in complex life cycle of protozoa remains largely unexplored. Here we have identified and characterized two phosphopantetheinyl transferases from D. discoideum that exhibit distinct functional specificity. DiAcpS specifically modifies a stand-alone acyl carrier protein (ACP) that possesses a mitochondrial import signal. DiSfp in contrast is specific to Type I multifunctional PKS/fatty acid synthase proteins and cannot modify the stand-alone ACP. The mRNA of two PPTases can be detected during the vegetative as well as starvation-induced developmental pathway and the disruption of either of these genes results in non-viable amoebae. Our studies show that both PPTases play an important role in Dictyostelium biology and provide insight into the importance of PPTases in lower eukaryotes.

Every polyketide synthase module has an acyl carrier protein (ACP) and a ketosynthase (KS) domain that collaborate to catalyze chain elongation. The same ACP then engages the KS domain of the next module to facilitate chain transfer. Understanding the mechanism for this orderly progress of the growing polyketide chain represents a fundamental challenge in assembly line enzymology. Using both experimental and computational approaches, the molecular basis for KS-ACP interactions in the 6-deoxyerythronolide B synthase has been decoded. Surprisingly, KS-ACP recognition is controlled at different interfaces during chain elongation versus chain transfer. In fact, chain elongation is controlled at a docking site remote from the catalytic center. Not only do our findings reveal a new principle in the modular control of polyketide antibiotic biosynthesis, they also provide a rationale for the mandatory homodimeric structure of polyketide synthases, in contrast to the monomeric nonribosomal peptide synthetases.

Bacterial acyl carrier protein (ACP) is a highly anionic, 9 kDa protein that functions as a cofactor protein in fatty acid biosynthesis. Escherichia coli ACP is folded at neutral pH and in the absence of divalent cations, while Vibrio harveyi ACP, which is very similar at 86% sequence identity, is unfolded under the same conditions. V. harveyi ACP adopts a folded conformation upon the addition of divalent cations such as Ca(2+) and Mg(2+) and a mutant, A75H, was previously identified that restores the folded conformation at pH 7 in the absence of divalent cations. In this study we sought to understand the unique folding behavior of V. harveyi ACP using NMR spectroscopy and biophysical methods. The NMR solution structure of V. harveyi ACP A75H displays the canonical ACP structure with four helices surrounding a hydrophobic core, with a narrow pocket closed off from the solvent to house the acyl chain. His-75, which is charged at neutral pH, participates in a stacking interaction with Tyr-71 in the far C-terminal end of helix IV. pH titrations and the electrostatic profile of ACP suggest that V. harveyi ACP is destabilized by anionic charge repulsion around helix II that can be partially neutralized by His-75 and is further reduced by divalent cation binding. This is supported by differential scanning calorimetry data which indicate that calcium binding further increases the melting temperature of V. harveyi ACP A75H by ∼20 °C. Divalent cation binding does not alter ACP dynamics on the ps-ns timescale as determined by (15)N NMR relaxation experiments, however, it clearly stabilizes the protein fold as observed by hydrogen-deuterium exchange studies. Finally, we demonstrate that the E. coli ACP H75A mutant is similarly unfolded as wild-type V. harveyi ACP, further stressing the importance of this particular residue for proper protein folding.

Serine palmitoyltransferase (SPT) catalyses the first step in the de novo biosynthesis of sphingolipids (SLs). It uses a decarboxylative Claisen-like condensation reaction to couple L-serine with palmitoyl-CoA to generate a long-chain base product, 3-ketodihydrosphingosine. SLs are produced by mammals, plants, yeast, and some bacteria, and we have exploited the complete genome sequence of Sphingomonas wittichii to begin a complete analysis of bacterial sphingolipid biosynthesis. Here, we describe the enzymatic characterization of the SPT from this organism and present its high-resolution x-ray structure. Moreover, we identified an open reading frame with high sequence homology to acyl carrier proteins (ACPs) that are common to fatty acid biosynthetic pathways. This small protein was co-expressed with the SPT and we isolated and characterised the apo- and holo-forms of the ACP. Our studies suggest a link between fatty acid and sphingolipid metabolism., (Copyright 2010 Wiley Periodicals, Inc.)

The pathways ensuring the efficient uptake of zinc are crucial for the ability of bacteria to multiply in the infected host. To better understand bacterial responses to zinc deficiency, we have investigated the role of the periplasmic protein ZinT in Salmonella enterica serovar Typhimurium. We have found that zinT expression is regulated by Zur and parallels that of ZnuA, the periplasmic component of the zinc transporter ZnuABC. Despite the fact that ZinT contributes to Salmonella growth in media containing little zinc, disruption of zinT does not significantly affect virulence in mice. The role of ZinT became clear using strains expressing a mutated form of ZnuA lacking a characteristic histidine-rich domain. In fact, Salmonella strains producing this modified form of ZnuA exhibited a ZinT-dependent capability to import zinc either in vitro or in infected mice, suggesting that ZinT and the histidine-rich region of ZnuA have redundant function. The hypothesis that ZinT and ZnuA cooperate in the process of zinc recruitment is supported by the observation that they form a stable binary complex in vitro. Although the presence of ZinT is not strictly required to ensure the functionality of the ZnuABC transporter, our data suggest that ZinT facilitates metal acquisition during severe zinc shortage.

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.

Acyl carrier proteins (ACPs) are required for the transfer of acyl intermediates during fatty acid and polyketide syntheses. In Sinorhizobium meliloti 1021 there are five known ACPs: AcpP, NodF, AcpXL, the ACP domain in RkpA and SMb20651. The genome sequence of S. meliloti 1021 also reveals the ORF SMc01553, annotated as a putative ACP. smc01553 is 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 in Escherichia coli was labelled in vivo with [(3)H]beta-alanine, a biosynthetic building block of the 4'-phosphopantetheine prosthetic group of ACPs. The purified SMc01553 was modified with 4'-phosphopantetheine in the presence of S. meliloti holo-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 from S. meliloti. Using RT-PCR we could show the presence of mRNA for SMc01553 and of the duplicated ORF SMb22007 in cultures of S. 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.

Bacteria respond to nutritional stress by producing (p)ppGpp, which triggers a stringent response resulting in growth arrest and expression of resistance genes. In Escherichia coli, RelA produces (p)ppGpp upon amino acid starvation by detecting stalled ribosomes. The SpoT enzyme responds to various other types of starvation by unknown mechanisms. We previously described an interaction between SpoT and the central cofactor of lipid synthesis, acyl carrier protein (ACP), which is involved in detecting starvation signals in lipid metabolism and triggering SpoT-dependent (p)ppGpp accumulation. However, most bacteria possess a unique protein homologous to RelA/SpoT (Rsh) that is able to synthesize and degrade (p)ppGpp and is therefore more closely related to SpoT function. In this study, we asked if the ACP-SpoT interaction is specific for bacteria containing two RelA and SpoT enzymes or if it is a general feature that is conserved in Rsh enzymes. By testing various combinations of SpoT, RelA, and Rsh enzymes and ACPs of E. coli, Pseudomonas aeruginosa, Bacillus subtilis and Streptococcus pneumoniae, we found that the interaction between (p)ppGpp synthases and ACP seemed to be restricted to SpoT proteins of bacteria containing the two RelA and SpoT proteins and to ACP proteins encoded by genes located in fatty acid synthesis operons. When Rsh enzymes from B. subtilis and S. pneumoniae are produced in E. coli, the behavior of these enzymes is different from the behavior of both RelA and SpoT proteins with respect to (p)ppGpp synthesis. This suggests that bacteria have evolved several different modes of (p)ppGpp regulation in order to respond to nutrient starvation.

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 of Sinorhizobium meliloti 1021 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 in Escherichia coli, [3H]beta-alanine, a biosynthetic building block of 4'-phosphopantetheine, was incorporated into the protein in vivo. The purified SMb20651 was modified with 4'-phosphopantetheine in the presence of S. meliloti holo-ACP synthase (AcpS). Also, holo-SMb20651 was modified in vitro with a malonyl group by malonyl CoA-ACP transacylase. In E. 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 in S. meliloti as holo-SMb20651 and acyl-SMb20651.

This work describes the isolation and characterization of an acyl carrier protein (ACP) mutant from Burkholderia cenocepacia J2315, a strain of the Burkholderia cepacia complex (Bcc). Bcc comprises at least 9 species that emerged as opportunistic pathogens able to cause life-threatening infections, particularly severe among cystic fibrosis patients. Bacterial ACPs are the donors of the acyl moiety involved in the biosynthesis of fatty acids, which play a central role in metabolism. The mutant was found to exhibit an increased ability to form biofilms in vitro, a more hydrophobic cell surface and reduced ability to colonize and kill the nematode Caenorhabditis elegans, used as a model of infection. The B. cenocepacia J2315 ACP protein is composed of 79 amino acid residues, with a predicted molecular mass and pI of 8.71kDa and 4.08, respectively. The ACP amino acid sequence was found to be 100% conserved within the genomes of the 52 Burkholderia strains sequenced so far. These data, together with results showing that the predicted structure of B. cenocepacia J2315 ACP is remarkably similar to the Escherichia coli AcpP, highlight its potential as a target to develop antibacterial agents to combat infections caused not only by Bcc species, but also by other Burkholderia species, especially B. pseudomallei and B. mallei.

We have cloned the nodFE operon from Sinorhizobium sp. strain MUS10. MUS10 NodF shows sequence homology to acyl carrier protein and enables an S. meliloti nodF mutant to effectively nodulate alfalfa. Our results demonstrate the occurrence of nodFE in a symbiont that nodulates a legume host not belonging to the galegoid group.

Electrospray ionization mass spectrometry (ESI-MS) can be used to monitor conformational changes of proteins in solution based on the charge state distribution (CSD) of the corresponding gas-phase ions, although relatively few studies of acidic proteins have been reported. Here, we have compared the CSD and solution structure of recombinant Vibrio harveyi acyl carrier protein (rACP), a small acidic protein whose secondary and tertiary structure can be manipulated by pH, fatty acylation, and site-directed mutagenesis. Circular dichroism and intrinsic fluorescence demonstrated that apo-rACP adopts a folded helical conformation in aqueous solution below pH 6 or in 50% acetonitrile/0.1% formic acid, but is unfolded at neutral and basic pH values. A rACP mutant, in which seven conserved acidic residues were replaced with their corresponding neutral amides, was folded over the entire pH range of 5 to 9. However, under the same solvent conditions, both wild type and mutant ACPs exhibited similar CSDs (6(+)-9(+) species) at all pH values. Covalent attachment of myristic acid to the phosphopantetheine prosthetic group of rACP, which is known to stabilize a folded conformation in solution, also had little influence on its CSD in either positive or negative ion modes. Overall, our results are consistent with ACP as a "natively unfolded" protein in a dynamic conformational equilibrium, which allows access to (de)protonation events during the electrospray process.

Acyl carrier protein (ACP), a small protein essential for bacterial growth and pathogenesis, interacts with diverse enzymes during the biosynthesis of fatty acids, phospholipids, and other specialized products such as lipid A. NMR and hydrodynamic studies have previously shown that divalent cations stabilize native helical ACP conformation by binding to conserved acidic residues at two sites (A and B) at either end of the "recognition" helix II. To examine the roles of these amino acids in ACP structure and function, site-directed mutagenesis was used to replace individual site A (Asp-30, Asp-35, Asp-38) and site B (Glu-47, Glu-53, Asp-56) residues in recombinant Vibrio harveyi ACP with the corresponding amides, along with combined mutations at each site (SA, SB) or both sites (SA/SB). Like native V. harveyi ACP, all individual mutants were unfolded at neutral pH but adopted a helical conformation in the presence of millimolar Mg(2+) or upon fatty acylation. Mg(2+) binding to sites A or B independently stabilized native ACP conformation, whereas mutant SA/SB was folded in the absence of Mg(2+), suggesting that charge neutralization is largely responsible for ACP stabilization by divalent cations. Asp-35 in site A was critical for holo-ACP synthase activity, while acyl-ACP synthetase and UDP-N-acetylglucosamine acyltransferase (LpxA) activities were more affected by mutations in site B. Both sites were required for fatty acid synthase activity. Overall, our results indicate that divalent cation binding site mutations have predicted effects on ACP conformation but unpredicted and variable consequences on ACP function with different enzymes.

Phosphopantetheinyl transferases (PPTases) are enzymes that catalyse the transfer of a 4'-phosphopantetheine moiety from CoA to a conserved serine residue of a carrier protein. These carrier proteins use the 4'-phosphopantetheine thiol to shuttle intermediates between the active sites of biosynthetic enzymes involved in fatty acid, non-ribosomal peptide and polyketide synthesis. Three PPTases have been previously been identified in Escherichia coli K-12 and other E. coli strains by homology searches and are encoded by the genes acpS, entD and acpT. Both AcpS and EntD have been well studied whereas the function of AcpT has been an enigma because no carrier protein substrate could be found. We report genetic and biochemical evidence that AcpT modifies two carrier proteins encoded in O-island 138, a cluster of fatty acid biosynthesis-like genes located adjacent to acpT in the genome of the pathogenic E. coli strain O157:H7 (E. coli K-12 and several other sequenced E. coli and Shigella strains lack O-island 138). The two carrier proteins of O-island 138 of strain O157:H7 are not modified (or only very poorly modified) by AcpS, the PPTase responsible for 4'-phosphopantetheine attachment to the acyl carrier protein (AcpP) of fatty acid synthesis. We demonstrate that AcpT cannot functionally replace AcpS in E. coli K-12 either in its native chromosomal location or upon insertion of acpT into the acpS chromosomal location. However, in the absence of AcpS activity AcpT does allow very slow growth thus providing a rationale for its retention in the absence of its cognate substrates. These results together with phylogenetic analyses and comparisons of the E. coli and Shigella strains of known genome sequence strongly argue that AcpT has been orphaned from its cognate substrates by a deletion event that occurred in a common ancestor of these organisms. This seems one of the few cases where a chromosomal rearrangement has been functionally demonstrated to be a deletion event rather than an insertion event in the reference organism. We also show that the previously reported suppression of an acpS mutation by the deletion of Lon protease is an artifact of the increased capsular polysaccharide production of lon strains.

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

Members of the Rhizobiaceae contain 27-hydroxyoctacosanoic acid (27OHC(28:0)) in their lipid A. A Rhizobium leguminosarum 3841 acpXL mutant (named here Rlv22) lacking a functional specialized acyl carrier lacked 27OHC(28:0) in its lipid A, had altered growth and physiological properties (e.g., it was unable to grow in the presence of an elevated salt concentration [0.5% NaCl]), and formed irregularly shaped bacteroids, and the synchronous division of this mutant and the host plant-derived symbiosome membrane was disrupted. In spite of these defects, the mutant was able to persist within the root nodule cells and eventually form, albeit inefficiently, nitrogen-fixing bacteroids. This result suggested that while it is in a host root nodule, the mutant may have some mechanism by which it adapts to the loss of 27OHC(28:0) from its lipid A. In order to further define the function of this fatty acyl residue, it was necessary to examine the lipid A isolated from mutant bacteroids. In this report we show that addition of 27OHC(28:0) to the lipid A of Rlv22 lipopolysaccharides is partially restored in Rlv22 acpXL mutant bacteroids. We hypothesize that R. leguminosarum bv. viciae 3841 contains an alternate mechanism (e.g., another acp gene) for the synthesis of 27OHC(28:0), which is activated when the bacteria are in the nodule environment, and that it is this alternative mechanism which functionally replaces acpXL and is responsible for the synthesis of 27OHC(28:0)-containing lipid A in the Rlv22 acpXL bacteroids.

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

Lipid A is the hydrophobic anchor of lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria. Lipid A of all Rhizobiaceae is acylated with a long fatty acid chain, 27-hydroxyoctacosanoic acid. Biosynthesis of this long acyl substitution requires a special acyl carrier protein, AcpXL, which serves as a donor of C28 (omega-1)-hydroxylated fatty acid for acylation of rhizobial lipid A (Brozek, K.A., Carlson, R.W., and Raetz, C. R. (1996) J. Biol. Chem. 271, 32126-32136). To determine the biological function of the C28 acylation of lipid A, we constructed an acpXL mutant of Sinorhizobium meliloti strain 1021. Gas-liquid chromatography and mass spectrometry analysis of the fatty acid composition showed that the acpXL mutation indeed blocked C28 acylation of lipid A. SDS-PAGE analysis of acpXL mutant LPS revealed only a fast migrating band, rough LPS, whereas the parental strain 1021 manifested both rough and smooth LPS. Regardless of this, the LPS of parental and mutant strains had a similar sugar composition and exposed the same antigenic epitopes, implying that different electrophoretic profiles might account for different aggregation properties of LPS molecules with and without a long acyl chain. The acpXL mutant of strain 1021 displayed sensitivity to deoxycholate, delayed nodulation of Medicago sativa, and a reduced competitive ability. However, nodules elicited by this mutant on roots of M. sativa and Medicago truncatula had a normal morphology and fixed nitrogen. Thus, the C28 fatty acid moiety of lipid A is not crucial, but it is beneficial for establishing an effective symbiosis with host plants. acpXL lies upstream from a cluster of five genes, including msbB (lpxXL), which might be also involved in biosynthesis and transfer of the C28 fatty acid to the lipid A precursor.

The structure of the lipid A from Rhizobium etli and Rhizobium leguminosarum lipopolysaccharides (LPSs) lacks phosphate and contains a galacturonosyl residue at its 4' position, an acylated 2-aminogluconate in place of the proximal glucosamine, and a very long chain omega-1 hydroxy fatty acid, 27-hydroxyoctacosanoic acid (27OHC28:0). The 27OHC28:0 moiety is common in lipid A's among members of the Rhizobiaceae and also among a number of the facultative intracellular pathogens that form chronic infections, e.g., Brucella abortus, Bartonella henselae, and Legionella pneumophila. In this paper, a mutant of R. leguminosarum was created by placing a kanamycin resistance cassette within acpXL, the gene which encodes the acyl carrier protein for 27OHC28:0. The result was an LPS containing a tetraacylated lipid A lacking 27OHC28:0. A small amount of the mutant lipid A may contain an added palmitic acid residue. The mutant is sensitive to changes in osmolarity and an increase in acidity, growth conditions that likely occur in the nodule microenvironment. In spite of the probably hostile microenvironment of the nodule, the acpXL mutant is still able to form nitrogen-fixing root nodules even though the appearance and development of nodules are delayed. Therefore, it is possible that the acpXL mutant has a host-inducible mechanism which enables it to adapt to these physiological changes.

The biosynthesis of epothilones, a family of hybrid polyketide (PK)/nonribosomal peptide (NRP) antitumor agents, provides an ideal system to study a hybrid PK/NRP natural product with significant biomedical value. Here the third enzyme involved in epothilone production, the five domain 195 kDa polyketide synthase (PKS) EpoC protein, has been expressed and purified from Escherichia coli. EpoC was combined with the first two enzymes of the epothilone biosynthesis pathway, the acyl carrier protein (ACP) domain of EpoA and EpoB, to reconstitute the early steps in epothilone biosynthesis. The acyltransferase (AT) domain of EpoC transfers the methylmalonyl moiety from methylmalonyl-CoA to the holo HS-acyl carrier protein (ACP) in an autoacylation reaction. The ketosynthase (KS) domain of EpoC decarboxylates the methylmalonyl-S-EpoC acyl enzyme to generate the carbon nucleophile that reacts with methylthiazolylcarboxyl-S-EpoB. The resulting condensation product can be reduced in the presence of NADPH by the ketoreductase (KR) domain of EpoC and then dehydrated by the dehydratase (DH) domain to produce the methylthiazolylmethylacrylyl-S-EpoC acyl enzyme intermediate that serves as the acyl donor for subsequent elongation of the epothilone chain. The acetyl-CoA donor can be replaced with propionyl-CoA, isobutyryl-CoA, and benzoyl-CoA and the acyl chains accepted by both EpoB and EpoC subunits to produce ethyl-, isopropyl-, and phenylthiazolylmethylacrylyl-S-EpoC acyl enzyme intermediates, suggesting that future combinatorial biosynthetic variations in epothilone assembly may be feasible. These results demonstrate in vitro reconstitution of both the PKS/NRPS interface (EpoA-ACP/B) and the NRPS/PKS interface (EpoB/C) in the assembly line for this antitumor natural product.

The HMWP2 subunit of yersiniabactin (Ybt) synthetase, a 230 kDa nonribosomal peptide synthetase (NRPS) making the N-terminus of the Ybt siderophore of Yersinia pestis, has one cysteine-specific adenylation (A) domain, three carrier protein domains (ArCP, PCP1, PCP2), and two heterocyclization domains (Cy1, Cy2). The A domain loads the two PCP domains with cysteines that get heterocyclized by the Cy domains to yield a tricyclic hydroxyphenylthiazolinylthiazolinyl (HPTT) chain lodged in thioester linkage to the PCP2 domain. The interdomain recognition by the Cy1 and Cy2 domains for the three carrier proteins was tested using inactivating mutations at the conserved serine that is phosphopantetheinylated in each carrier domain (S52A, S1439A, and S1977A). These mutant forms of HMWP2 were tested for in trans complementation by carrier protein fragments: holo-ArCPs (S52A), holo-PCP1 and analogues (S1439A), and holo-PCP2 and analogues (S1977A). The S52A mutant tests the recognition of the Cy1 domain for donor acyl-ArCP substrates, while the S1439A mutant tests the specificity of the same Cy1 domain for downstream substrates presented by distinct PCPs. The S1439A likewise tests the recognition of Cy2 for its upstream PCP-tethered acyl donor. The S1977A mutant analogously tests the Cy2 domain for downstream Cys-PCP recognition. In all cases in trans complementation was successful with the carrier protein fragments, allowing kinetic probes of catalytic efficiency for PCP scaffolds and for uncoupling of the condensation and heterocyclization functions of Cy1 and Cy2. Overall, the Cy domains tested showed a definite selectivity for the upstream protein scaffold but were more relaxed toward the downstream acceptor protein. This work points to the importance of protein-protein interactions in mediating directional chain growth in NRPS and presents the first systematic exploration of how the protein scaffolds affect catalytic efficiency.

A genomic region containing the fatty acid biosynthetic (fab) genes was isolated from the sugarcane leaf-scald pathogen Xanthomonas albilineans. The order and predicted products of fabG (beta-ketoacyl reductase), acpP (acyl carrier protein), fabF (ketoacyl synthase II) and downstream genes in X. albilineans are very similar to those in Escherichia coli, with one exception. Sequence analysis, confirmed by insertional knockout and specific substrate feeding experiments, shows that the position occupied by pabC (encoding aminodeoxychorismate lyase) in other bacteria is occupied instead by pabB (encoding aminodeoxychorismate synthase component I) in X. albilineans. Downstream of pabB, X. albilineans resumes the arrangement common to characterized Gram-negative bacteria, with three transcriptionally coupled genes, encoding an ORF340 protein of undefined function, thymidylate kinase and delta' subunit of DNA polymerase III holoenzyme (HolB). Different species may obtain a common advantage from coordinated regulation of the same biosynthetic pathways using different genes in this region.

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

In rhizobia, besides the constitutive acyl carrier protein (AcpP) involved in the biosynthesis and transfer of common fatty acids, there are at least three specialized acyl carrier proteins (ACPs): (1) the flavonoid-inducible nodulation protein NodF; (2) the RkpF protein, which is required for the biosynthesis of rhizobial capsular polysaccharides; and (3) AcpXL, which transfers 27-hydroxyoctacosanoic acid to a sugar backbone during lipid A biosynthesis. Whereas the nucleotide sequences encoding the three specialized ACPs are known, only the amino acid sequence of the AcpP of Sinorhizobium meliloti was available. In this study, using reverse genetics, the genes for the constitutive AcpPs of S. meliloti and of Rhizobium leguminosarum were cloned and sequenced. Previously, it had been shown that NodF and RkpF can be overproduced in Escherichia coli using the T7 polymerase expression system. Using the same system, the constitutive AcpPs of S. meliloti and of R. leguminosarum, together with the specialized ACP AcpXL, were overproduced and purified. All the known ACPs of rhizobia can be labelled in vivo during expression in E. coli with radioactive beta-alanine added to the growth medium due to their modification with a 4'-phosphopantetheine prosthetic group. The availability of all functionally different ACPs should help to unravel how different fatty acids are targeted towards different biosynthetic pathways in one organism.

The 110 kDa haemolysin protoxin (proHlyA) is activated in the Escherichia coli cytosol by acyl carrier protein-dependent fatty acylation of two internal lysine residues, directed by the co-synthesized protein HlyC. Using an in vitro maturation reaction containing purified protoxin peptides and acylACP, we show unambiguously that HlyC possesses an apparently unique acyltransferase activity fully described by Michaelis-Menten analysis. The Vmax of HlyC at saturating levels of both substrates was approximately 115 nmol acyl group min-1 mg-1 with KMacylACP of 260 nM and KMproHlyA of 27 nM, kinetic parameters sufficient to explain why in vivo HlyC is required at a concentration equimolar to proHlyA. HlyC bound the fatty acyl group from acylACP to generate an acylated HlyC intermediate that was depleted in the presence of proHlyA, but enriched in the presence of proHlyA derivatives lacking acylation target sites. HlyC was also able to bind in vivo 4'-phosphopantetheine. Substitution of conserved amino acids that could act as putative covalent attachment sites did not prevent binding of the fatty acyl or 4'-phosphopantetheine groups. These data and substrate variation analyses suggest that the unique acylation reaction does not involve covalent attachment of fatty acid to the acyltransferase, but rather that it proceeds via a sequential ordered Bi-Bi reaction mechanism, requiring the formation of a non-covalent ternary acylACP-HlyC-proHlyA complex.

Background: Polyketide synthases (PKSs) are bacterial multienzyme systems that synthesize a broad range of natural products. The 'minimal' PKS consists of a ketosynthase, a chain length factor, an acyl carrier protein and a malonyl transferase. Auxiliary components (ketoreductases, aromatases and cyclases are involved in controlling the oxidation level and cyclization of the nascent polyketide chain. We describe the heterologous expression and reconstitution of several auxiliary PKS components including the actinorhodin ketoreductase (act KR), the griseusin aromatase/cyclase (gris ARO/CYC), and the tetracenomycin aromatase/cyclase (tcm ARO/CYC)., Results: The polyketide products of reconstituted act and tcm PKSs were identical to those identified in previous in vivo studies. Although stable protein-protein interactions were not detected between minimal and auxiliary PKS components, kinetic analysis revealed that the extended PKS comprised of the act minimal PKS, the act KR and the gris ARO/CYC had a higher turnover number than the act minimal PKS plus the act KR or the act minimal PKS alone. Adding the tcm ARO/CYC to the tcm minimal PKS also increased the overall rate., Conclusions: Until recently the principal strategy for functional analysis of PKS subunits was through heterologous expression of recombinant PKSs in Streptomyces. Our results corroborate the implicit assumption that the product isolated from whole-cell systems is the dominant product of the PKS. They also suggest that an intermediate is channeled between the various subunits, and pave the way for more detailed structural and mechanistic analysis of these multienzyme systems.

A DNA fragment containing the Pseudomonas aeruginosa fabD (encoding malonyl-coenzyme A [CoA]:acyl carrier protein [ACP] transacylase), fabG (encoding beta-ketoacyl-ACP reductase), acpP (encoding ACP), and fabF (encoding beta-ketoacyl-ACP synthase II) genes was cloned and sequenced. This fab gene cluster is delimited by the plsX (encoding a poorly understood enzyme of phospholipid metabolism) and pabC (encoding 4-amino-4-deoxychorismate lyase) genes; the fabF and pabC genes seem to be translationally coupled. The fabH gene (encoding beta-ketoacyl-ACP synthase III), which in most gram-negative bacteria is located between plsX and fabD, is absent from this gene cluster. A chromosomal temperature-sensitive fabD mutant was obtained by site-directed mutagenesis that resulted in a W258Q change. A chromosomal fabF insertion mutant was generated, and the resulting mutant strain contained substantially reduced levels of cis-vaccenic acid. Multiple attempts aimed at disruption of the chromosomal fabG gene were unsuccessful. We purified FabD as a hexahistidine fusion protein (H6-FabD) and ACP in its native form via an ACP-intein-chitin binding domain fusion protein, using a novel expression and purification scheme that should be applicable to ACP from other bacteria. Matrix-assisted laser desorption-ionization spectroscopy, native polyacrylamide electrophoresis, and amino-terminal sequencing revealed that (i) most of the purified ACP was properly modified with its 4'-phosphopantetheine functional group, (ii) it was not acylated, and (iii) the amino-terminal methionine was removed. In an in vitro system, purified ACP functioned as acyl acceptor and H(6)-FabD exhibited malonyl-CoA:ACP transacylase activity.