Ligand binding induces an ammonia channel in 2-amino-2-desoxyisochorismate (ADIC) synthase PhzE.
ABSTRACT: PhzE utilizes chorismate and glutamine to synthesize 2-amino-2-desoxyisochorismate (ADIC) in the first step of phenazine biosynthesis. The PhzE monomer contains both a chorismate-converting menaquinone, siderophore, tryptophan biosynthesis (MST) and a type 1 glutamine amidotransferase (GATase1) domain connected by a 45-residue linker. We present here the crystal structure of PhzE from Burkholderia lata 383 in a ligand-free open and ligand-bound closed conformation at 2.9 and 2.1 Å resolution, respectively. PhzE arranges in an intertwined dimer such that the GATase1 domain of one chain provides NH(3) to the MST domain of the other. This quaternary structure was confirmed by small angle x-ray scattering. Binding of chorismic acid, which was found converted to benzoate and pyruvate in the MST active centers of the closed form, leads to structural rearrangements that establish an ammonia transport channel approximately 25 Å in length within each of the two MST/GATase1 functional units of the dimer. The assignment of PhzE as an ADIC synthase was confirmed by mass spectrometric analysis of the product, which was also visualized at 1.9 Å resolution by trapping in crystals of an inactive mutant of PhzD, an isochorismatase that catalyzes the subsequent step in phenazine biosynthesis. Unlike in some of the related anthranilate synthases, no allosteric inhibition was observed in PhzE. This can be attributed to a tryptophan residue of the protein blocking the potential regulatory site. Additional electron density in the GATase1 active center was identified as zinc, and it was demonstrated that Zn(2+), Mn(2+), and Ni(2+) reduce the activity of PhzE.
Project description:The chloramphenicol producer Streptomyces venezuelae contains an enzyme, SvTrpEG, that has a high degree of amino acid sequence similarity to the phenazine biosynthetic enzyme PhzE of certain species of Pseudomonas. PhzE has the sequence signature of an anthranilate synthase, but recent evidence indicates that it catalyzes the production of 2-amino-2-deoxyisochorismate [corrected] (ADIC), an intermediate in the two-step anthranilate synthase reaction, not anthranilate. In order to determine if SvTrpEG is likewise an ADIC synthase, we have cloned the gene for SvTrpEG, expressed the recombinant enzyme in Escherichia coli, and purified the enzyme. Analysis of the SvTrpEG-catalyzed reaction mixture using UV-visible spectrophotometry, fluorescence spectrometry, and high-performance liquid chromatography shows that the product of the reaction is anthranilate, not ADIC. Our results therefore reveal that, despite its sequence similarity to PhzE, SvTrpEG is an anthranilate synthase, not an ADIC synthase.
Project description:A fast and efficient approach was established to identify bacteria possessing the potential to biosynthesize phenazines, which are of special interest regarding their antimicrobial activities. Sequences of phzE genes, which are part of the phenazine biosynthetic pathway, were used to design one universal primer system and to analyze the ability of bacteria to produce phenazine. Diverse bacteria from different marine habitats and belonging to six major phylogenetic lines were investigated. Bacteria exhibiting phzE gene fragments affiliated to Firmicutes, Alpha- and Gammaproteobacteria, and Actinobacteria. Thus, these are the first primers for amplifying gene fragments from Firmicutes and Alphaproteobacteria. The genetic potential for phenazine production was shown for four type strains belonging to the genera Streptomyces and Pseudomonas as well as for 13 environmental isolates from marine habitats. For the first time, the genetic ability of phenazine biosynthesis was verified by analyzing the metabolite pattern of all PCR-positive strains via HPLC-UV/MS. Phenazine production was demonstrated for the type strains known to produce endophenazines, 2-hydroxy-phenazine, phenazine-1-carboxylic acid, phenazine-1,6-dicarboxylic acid, and chlororaphin as well as for members of marine Actinobacteria. Interestingly, a number of unidentified phenazines possibly represent new phenazine structures.
Project description:One of the fundamental questions of enzymology is how catalytic power is derived. This review focuses on recent developments in the structure--function relationships of chorismate-utilizing enzymes involved in siderophore biosynthesis to provide insight into the biocatalysis of pericyclic reactions. Specifically, salicylate synthesis by the two-enzyme pathway in Pseudomonas aeruginosa is examined. The isochorismate-pyruvate lyase is discussed in the context of its homologues, the chorismate mutases, and the isochorismate synthase is compared to its homologues in the MST family (menaquinone, siderophore, or tryptophan biosynthesis) of enzymes. The tentative conclusion is that the activities observed cannot be reconciled by inspection of the active site participants alone. Instead, individual activities must arise from unique dynamic properties of each enzyme that are tuned to promote specific chemistries.
Project description:The shikimate pathway of bacteria, fungi, and plants generates chorismate, which is drawn into biosynthetic pathways that form aromatic amino acids and other important metabolites, including folates, menaquinone, and siderophores. Many of the pathways initiated at this branch point transform chorismate using an MST enzyme. The MST enzymes (menaquinone, siderophore, and tryptophan biosynthetic enzymes) are structurally homologous and magnesium-dependent, and all perform similar chemical permutations to chorismate by nucleophilic addition (hydroxyl or amine) at the 2-position of the ring, inducing displacement of the 4-hydroxyl. The isomerase enzymes release isochorismate or aminodeoxychorismate as the product, while the synthase enzymes also have lyase activity that displaces pyruvate to form either salicylate or anthranilate. This has led to the hypothesis that the isomerase and lyase activities performed by the MST enzymes are functionally conserved. Here we have developed tailored pre-steady-state approaches to establish the kinetic mechanisms of the isochorismate and salicylate synthase enzymes of siderophore biosynthesis. Our data are centered on the role of magnesium ions, which inhibit the isochorismate synthase enzymes but not the salicylate synthase enzymes. Prior structural data have suggested that binding of the metal ion occludes access or egress of substrates. Our kinetic data indicate that for the production of isochorismate, a high magnesium ion concentration suppresses the rate of release of product, accounting for the observed inhibition and establishing the basis of the ordered-addition kinetic mechanism. Moreover, we show that isochorismate is channeled through the synthase reaction as an intermediate that is retained in the active site by the magnesium ion. Indeed, the lyase-active enzyme has 3 orders of magnitude higher affinity for the isochorismate complex relative to the chorismate complex. Apparent negative-feedback inhibition by ferrous ions is documented at nanomolar concentrations, which is a potentially physiologically relevant mode of regulation for siderophore biosynthesis in vivo.
Project description:<h4>Abstract</h4>In this contribution, we report synthetic strategies towards potential ligands for the study of binding differences between PhzE, the first enzyme in the biosynthesis of phenazines, and the related enzyme anthranilate synthase. The ligands were designed with the overriding goal to develop new antibiotics via downregulation of phenazine biosynthesis.<h4>Graphical abstract</h4>
Project description:Pathogenic enterobacteria need to survive the extreme acidity of the stomach to successfully colonize the human gut. Enteric bacteria circumvent the gastric acid barrier by activating extreme acid-resistance responses, such as the arginine-dependent acid resistance system. In this response, l-arginine is decarboxylated to agmatine, thereby consuming one proton from the cytoplasm. In Escherichia coli, the l-arginine/agmatine antiporter AdiC facilitates the export of agmatine in exchange of l-arginine, thus providing substrates for further removal of protons from the cytoplasm and balancing the intracellular pH. We have solved the crystal structures of wild-type AdiC in the presence and absence of the substrate agmatine at 2.6-Å and 2.2-Å resolution, respectively. The high-resolution structures made possible the identification of crucial water molecules in the substrate-binding sites, unveiling their functional roles for agmatine release and structure stabilization, which was further corroborated by molecular dynamics simulations. Structural analysis combined with site-directed mutagenesis and the scintillation proximity radioligand binding assay improved our understanding of substrate binding and specificity of the wild-type l-arginine/agmatine antiporter AdiC. Finally, we present a potential mechanism for conformational changes of the AdiC transport cycle involved in the release of agmatine into the periplasmic space of E. coli.
Project description:The Erd?s-Rényi (ER) random graph G(n, p) analytically characterizes the behaviors in complex networks. However, attempts to fit real-world observations need more sophisticated structures (e.g., multilayer networks), rules (e.g., Achlioptas processes), and projections onto geometric, social, or geographic spaces. The p-adic number system offers a natural representation of hierarchical organization of complex networks. The p-adic random graph interprets n as the cardinality of a set of p-adic numbers. Constructing a vast space of hierarchical structures is equivalent for combining number sequences. Although the giant component is vital in dynamic evolution of networks, the structure of multiple big components is also essential. Fitting the sizes of the few largest components to empirical data was rarely demonstrated. The p-adic ultrametric enables the ER model to simulate multiple big components from the observations of genetic interaction networks, social networks, and epidemics. Community structures lead to multimodal distributions of the big component sizes in networks, which have important implications in intervention of spreading processes.
Project description:In Pseudomonas aeruginosa (Pae), the shikimate pathway end product, chorismate, serves as the last common precursor for the biosynthesis of both primary aromatic metabolites, including phenylalanine, tyrosine and tryptophan, and secondary aromatic metabolites, including phenazine-1-carboxylic acid (PCA) and pyocyanin (PYO). The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first committed step of the shikimate pathway, en route to chorismate. P. aeruginosa expresses multiple, distinct DAH7PSs that are associated with either primary or secondary aromatic compound biosynthesis. Here we report the structure of a type II DAH7PS, encoded by phzC as part of the duplicated phenazine biosynthetic cluster, from P. aeruginosa (PAO1) revealing for the first time the structure of a type II DAH7PS involved in secondary metabolism. The omission of the structural elements α2a and α2b, relative to other characterised type II DAH7PSs, leads to the formation of an alternative, dimeric, solution-state structure for this type II DAH7PS with an oligomeric interface that has not previously been characterised and that does not facilitate the formation of aromatic amino acid allosteric binding sites. The sequence similarity and, in particular, the common N-terminal extension suggest a common origin for the type II DAH7PSs from P. aeruginosa. The results described in the present study support an expanded classification of the type II DAH7PSs as type IIA and type IIB based on sequence characteristics, structure and function of the resultant proteins, and on defined physiological roles within primary or secondary metabolism.
Project description:Natural pyrrolobenzodiazepines (PBDs) form a large and structurally diverse group of antitumour microbial metabolites produced through complex pathways, which are encoded within biosynthetic gene clusters. We sequenced the gene cluster of limazepines and proposed their biosynthetic pathway based on comparison with five available gene clusters for the biosynthesis of other PBDs. Furthermore, we tested two recombinant proteins from limazepine biosynthesis, Lim5 and Lim6, with the expected substrates in vitro. The reactions monitored by LC-MS revealed that limazepine biosynthesis involves a new way of 3-hydroxyanthranilic acid formation, which we refer to as the chorismate/DHHA pathway and which represents an alternative to the kynurenine pathway employed for the formation of the same precursor in the biosynthesis of other PBDs. The chorismate/DHHA pathway is presumably also involved in the biosynthesis of PBD tilivalline, several natural products unrelated to PBDs, and its part is shared also with phenazine biosynthesis. The similarities between limazepine and phenazine biosynthesis indicate tight evolutionary links between these groups of compounds.
Project description:The arginine-agmatine antiporter (AdiC) is a component of an acid resistance system developed by enteric bacteria to resist gastric acidity. In order to avoid neutral proton antiport, the monovalent form of arginine, about as abundant as its divalent form under acidic conditions, should be selectively bound by AdiC for transport into the cytosol. In this study, we shed light on the mechanism through which AdiC distinguishes Arg+ from Arg2+ of arginine by investigating the binding of both forms in addition to that of divalent agmatine, using a combination of molecular dynamics simulations with molecular and quantum mechanics calculations. We show that AdiC indeed preferentially binds Arg+. The weaker binding of divalent compounds results mostly from their greater tendency to remain hydrated than Arg+. Our data suggests that the binding of Arg+ promotes the deprotonation of Glu208, a gating residue, which in turn reinforces its interactions with AdiC, leading to longer residence times of Arg+ in the binding site. Although the total electric charge of the ligand appears to be the determinant factor in the discrimination process, two local interactions formed with Trp293, another gating residue of the binding site, also contribute to the selection mechanism: a cation-? interaction with the guanidinium group of Arg+ and an anion-? interaction involving Glu208.