We determined the crystal structure of Thr1, the self-standing adenylation domain involved in the nonribosomal-like biosynthesis of free 4-chlorothreonine in Streptomyces sp. OH-5093. Thr1 shows two monomers in the crystallographic asymmetric unit with different relative orientations of the C- and N-terminal subdomains both in the presence of substrates and in the unliganded form. Cocrystallization with substrates, adenosine 5'-triphosphate and l-threonine, yielded one monomer containing the two substrates and the other in complex with l-threonine adenylate, locked in a postadenylation state. Steady-state kinetics showed that Thr1 activates l-Thr and its stereoisomers, as well as d-Ala, l- and d-Ser, albeit with lower efficiency. Modeling of these substrates in the active site highlighted the molecular bases of substrate discrimination. This work provides the first crystal structure of a threonine-activating adenylation enzyme, a contribution to the studies on conformational rearrangement in adenylation domains and on substrate recognition in nonribosomal biosynthesis. DATABASE:Structural data are available in the Protein Data Bank under the accession number 5N9W and 5N9X.

Background Pyridoxal 5′-phosphate (PLP)-dependent enzymes of fold type I, the most studied structural class of the PLP-dependent enzyme superfamily, are known to exist as stand-alone homodimers or homotetramers. These enzymes have been found also embedded in multimodular and multidomain assembly lines involved in the biosynthesis of polyketides (PKS) and nonribosomal peptides (NRPS). The aim of this work is to provide a proteome-wide view of the distribution and characteristics of type I domains covalently integrated in these assemblies in prokaryotes. Results An ad-hoc Hidden Markov profile was calculated using a sequence alignment derived from a multiple structural superposition of distantly related PLP-enzymes of fold type I. The profile was utilized to scan the sequence databank and to collect the proteins containing at least one type I domain linked to a component of an assembly line in bacterial genomes. The domains adjacent to a carrier protein were further investigated. Phylogenetic analysis suggested the presence of four PLP-dependent families: Aminotran_3, Beta_elim_lyase and Pyridoxal_deC, occurring mainly within mixed NRPS/PKS clusters, and Aminotran_1_2 found mainly in PKS clusters. Sequence similarity to the reference PLP enzymes with solved structures ranged from 24 to 42% identity. Homology models were built for each representative type I domain and molecular docking simulations with putative substrates were carried out. Prediction of the protein-protein interaction sites evidenced that the surface regions of the type I domains embedded within multienzyme assemblies were different from those of the self-standing enzymes; these structural features appear to be required for productive interactions with the adjacent domains in a multidomain context. Conclusions This work provides a systematic view of the occurrence of type I domain within NRPS and PKS assembly lines and it predicts their structural characteristics using computational methods. Comparison with the corresponding stand-alone enzymes highlighted the common and different traits related to various aspects of their structure-function relationship. Therefore, the results of this work, on one hand contribute to the understanding of the functional and structural diversity of the PLP-dependent type I enzymes and, on the other, pave the way to further studies aimed at their applications in combinatorial biosynthesis.

Molecular cloning of the biosynthetic gene cluster involved in the production of free 4‐chlorothreonine in Streptomyces sp. OH‐5093 showed the presence of six ORFs: thr1, thr2, thr3, orf1, orf2 and thr4. According to bioinformatic analysis, thr1, thr2, thr3 and thr4 encode a free‐standing adenylation domain, a carrier protein, an Fe(II) nonheme α‐ketoglutarate‐dependent halogenase and a thioesterase, respectively, indicating the role of these genes in the activation and halogenation of threonine and the release of 4‐chlorothreonine in a pathway closely reflecting the formation of this amino acid in the biosynthesis of the lipodepsipeptide syringomycin from Pseudomonas syringae pv. syringae B301DR. Orf1 and orf2 show sequence similarity with alanyl/threonyl‐tRNA synthetases editing domains and drug metabolite transporters, respectively. We show that thr3 can replace the halogenase gene syrB2 in the biosynthesis of syringomycin, by functional complementation of the mutant P. s. pv. syringae strain BR135A1 inactivated in syrB2. We also provide an insight into the structure–function relationship of halogenases Thr3 and SyrB2 using homology modelling and site‐directed mutagenesis.DatabaseNucleotide sequence data have been deposited in the ebi.ac.uk/EMBL/GenBank databases under accession numbers: thr1 (gi:378781338|CCF23454); thr2 (gi:378781340|CCF23456); thr3 (gi:378781341|CCF23457); orf1 (gi:378781342|CCF23458); orf2 (gi:378781343|CCF23459); thr4 (gi:378781344|CCF23460)

Syringomycin E (SRE) is a member of a family of lipodepsipeptides that characterize the secondary metabolism of the plant-associated bacteria Pseudomonas syringae pv. syringae. It displays phytotoxic, antifungal and haemolytic activities, due to the membrane interaction and ion channel formation. To gain an insight into the conformation of SRE in the membrane environment, we studied the conformation of SRE bound to SDS micelle, a suitable model for the membrane-bound SRE. In fact, highly similar circular dichroism (CD) spectra were obtained for SRE bound to sodium dodecylsulphate (SDS) and to a phospholipid bilayer, indicating the conformational equivalence of SRE in these two media, at difference with the CD spectrum of SRE in water solution. The structure of SDS-bound SRE was determined by NMR spectroscopy combined with molecular dynamics calculations in octane environment. The results of this study highlight the influence of the interaction with lipids in determining the three-dimensional structure of SRE and provide the basis for further investigations on structural determinants of syringomycin E-membrane interaction.

There are some concerns about the use of Pseudomonas-based products as biocontrol agents because of the hemolytic activity shown by their metabolites. The effects of Pseudomonas lipodepsipeptides (LDPs) on mammals via ingestion and the LDP degradation during the digestion and intestinal permeability have not been evaluated. In this research, the susceptibility of different LDPs to degradation was assayed with enzymatic gastrointestinal digestion, and intestinal permeability to LDPs was investigated in an in vitro system based on an intestinal cell layer system. Results demonstrated that trypsin and chymotrypsin hydrolyze up to 50% of the various LDPs, and that proteolysis was further increased by pronase E treatment. A decrease in LDP hemolytic activity matched LDP degradation during the various steps of the digestion process. Moreover, it was shown that syringomycin E (SRE), the main known LDP, was not able to cross the intestinal cell layer, suggesting that SRE does not reach the bloodstream in vivo. It was concluded that the Pseudomonas-based biocontrol products do not represent a serious risk for consumer health. In fact, LDPs possibly present on biocontrol-treated agricultural commodities would likely be partially digested by gastrointestinal enzymes and would not be absorbed at the intestinal level.

The production of secondary metabolite lipopeptides by ice-nucleating Pseudomonas syringae strain 31R1 was investigated. Pseudomonas syringae strain 31R1 is a rifampicin-resistant derivative of P. syringae no. 31 used for the commercial production of snow. It is shown that P. syringae strain 31R1 produces antifungal lipodepsipeptides, syringomycins E and G, and, in addition, a novel and unique lipopeptide, peptin31. Spectroscopic and spectrometric analyses revealed that peptin31 is a linear undecalipopeptide with sequence identities to N- and C-terminal portions but lacking 11 amino acids of known lipodepsipeptide syringopeptin SPPhv. Peptin31 displayed antifungal activities against Rhodotorula pilimanae, Rhizoctonia solani, and Trichoderma harzianum and also hemolytic and antibacterial activities. Extracts of P. syringae strain 31R1 grown in medium with chloride were fungicidal, but not when grown without chloride. The latter extracts lacked peptin 31 and contained des-chloro forms of syringomycins E and G with low antifungal activities. Thus, the three lipopeptides account for the fungicidal properties of P. syringae 31R1 extracts. The occurrence of these bioactive metabolites should be considered when P. syringae no. 31 and its derivatives are used in products for making artificial snow.

SyrC, a component of the multienzyme system of syringomycin biosynthesis, has been shown to shuttle Thr/4-Cl-Thr between the thiolation domains SyrB1-T1 and SyrE-T8,9 by transiently linking it to Cys224 in the enzyme active site. We present data on the structure-function relationship in vivo of this protein and an in silico model of its three-dimensional structure. The biosynthetic activity of SyrC was not influenced when either Asp348 or His376 that together with Cys224 form a putative catalytic triad, were replaced with Ala, but it was abolished by the exchange Cys224 with Ser. The presence of the FLAG peptide on either the N- or C-terminus of the protein did not affect activity, whereas the deletion of the first 16 amino acids at the N-terminus or the insertion of Maltose Binding Protein abolished the production of syringomycin. We present the model of the three-dimensional structure of SyrC suggesting a homodimeric structure for the protein and biochemical data that are supportive of this model.

This work presents a computational analysis of the molecular characteristics shared by the adenylation domains from traditional nonribosomal peptide synthetases (NRPSs) and the group of the freestanding homologous enzymes: α‐aminoadipate semialdehyde dehydrogenase, α‐aminoadipate reductase and the protein Ebony. The results of systematic sequence comparisons allow us to conclude that a specificity‐conferring code, similar to that described for the NRPSs, can be recognized in such enzymes. The structural and functional roles of the residues involved in the substrate selection and binding are proposed through the analysis of the predicted interactions of the model active sites and their respective substrates. The indications deriving from this study can be useful for the programming of experiments aimed at a better characterization and at the engineering of this emerging group of single NRPS modules that are responsible for amino acid selection, activation and modification in the absence of other NRPS assembly line components.

The syringopeptins are a group of antimicrobial cyclic lipodepsipeptides produced by several plant-associated pseudomonads. A novel syringopeptin, SP508, was shown to be produced as two homologs (A and B) by Pseudomonas syringae pv. lachrymans strain 508 from apple and to structurally resemble syringopeptin SP22. SP508 differed from SP22 and other syringopeptins by having three instead of four alpha,beta-unsaturated amino acids and a longer beta-hydroxy acyl chain. Both SP508 and SP22 displayed growth-inhibitory activities against Mycobacterium smegmatis, other gram-positive bacteria, and yeasts but not against gram-negative bacteria. Structure-activity analyses of the SP508 and SP22 homologs indicated chemical structural features that lead to enhanced antimycobacterial activity by these pseudomonad cyclic lipodepsipeptides.

Lipodepsipeptides (LPDs) are a group of cyclic, acylated peptides produced by several Pseudomonas species. They are usually divided in two groups, mycins and peptins, on the basis of the size of the amino acidic part of the molecule. Mycins have a ring of 9 amino acids closed between the first and the last residue, peptins contain a more complex peptide moiety of up to 25 amino acids, partially cyclized. Both mycins and peptins attack the plasma membrane, but may have different target organisms. Comparing the mode of action of these two classes of LDPs on natural and model membranes we observed that all peptides induced red blood cell haemolysis and leakage of tonoplasts and liposornes by the formation of pores. The haemolytic activity of the smaller mycins was higher than that of the bigger peptins and proportional to the amphipathic index of the molecule. The extent of permeabilization was dependent also on the composition of the lipid membrane. In particular, mycins show a preference for sterols, whereas peptins are more active on phospholipids, especially sphingomyelin. These differences may have physiological implications. The formation of discrete ion channels, with anionic selectivity, was directly demonstrated by electrophysiological experiments performed on planar lipid bilayers or sugar beet vacuoles. The channels show sub-states and their properties in vacuoles and in planar lipid membranes were remarkably similar.

Structural features and biological activities of lipodepsipeptides produced by various strains of the widely spread phytopathogenic bacterium Pseudomonas syringae pv. syringae are presented here. Emphasis is put on biotechnological and potential biomedical applications of this family of secondary metabolites. In this context, structurally related metabolites from other Pseudomonas species are also considered.

Syringomycin E (SRE), a lipodepsinonapeptide produced by many Pseudomonas syringae pv. syringae strains, displays a prominent antifungal and haemolytic activity. The former is already exploited for post-harvest biocontrol of fruit and appears interesting in the perspective of medical applications; the latter hampers the use of this compound as a systemic drug. The ability to interact with biological membranes and form pores is at the basis of its biological activity. To gain insight into the structural features which are important for this process, we produced structural analogues of the metabolite and evaluated their activity on red blood cells and on artificial membranes of different compositions. The substitution of chlorine with hydrogen in the G terminal amino acid caused a marked decrease in the pore-forming activity, in accordance with the previously observed trend in the antifungal activity assay. The cooperativity of the process is not affected, but the activity of the deschloro-SRE analogue (SREH) at difference from SRE, is not influenced by the presence of sterols in the artificial lipid bilayers. The residual activity of SREH is abolished when the lactone ring is hydrolysed. Thus, both the presence of chlorine in the C-terminal residue and the integrity of the macrocycle appear to be important for the pore-forming activity of SRE.

Pseudomonas syringae pv. syringae produces two classes of pore-forming lipodepsipeptide phytotoxins that target host plasma membranes. Both syringomycin and syringopeptin are synthesised by modular nonribosomal peptide synthetases. The syringomycin (syr) and syringopeptin (syp) gene clusters are located adjacent to one another on the chromosome and are estimated to be 55 kb and 90 kb in size, respectively. Here we describe the functional organisation of the clusters, along with flanking DNA regions, which altogether cover an approximately 155-kb region of the genome. The predominant feature of the toxin clusters is the occurrence of peptide synthetase genes for syringomycin (i.e., syrB1 and syrE) and syringopeptin (i.e., sypA, sypB, and sypC). Associated with the toxin clusters are genes that are predicted to function in toxin secretion and regulation of biosynthesis genes. Progress in characterising functions of specific genes of the syr-syp cluster dedicated to syringomycin, syringopeptin, or both toxins is summarised.

The three-dimensional structures of the adenylation domains (A) of eight modules of SyrE, a peptide synthetase involved in the biosynthesis of syringomycin, was investigated by homology modeling using as a template the adenylation domain of gramicidin synthetase 1. Multiple sequence alignment of the adenylation domains of each of the eight modules of SyrE, allowed to identify the residues which delimitate the active site pockets. The active sites of the A domains of SyrE1 and SyrE2, involved in the insertion of the two serine residues in syringomycin, are essentially equivalent. The docking of these models with L-Ser and D-Ser showed that L-Ser is preferred as a substrate on the basis of stabilizing interactions. This is in accordance with previously shown stereo-specificity of these modules. Analogous investigation carried out on the modules SyrE3 and SyrE4 showed the preference for the L-isomer of their substrate, 2,4-diaminobutyric acid. In addition, the model of SyrE8, whose substrate amino acid is arginine, allowed to evidence specificity - conferring residues for this residue, which had not been previously reported.

Two strains (B728a and Y37) of the phytopathogenic bacterium Pseudomonas syringae pv. syringae isolated from bean (Phaseolus vulgaris) plants were shown to produce in culture both syringomycin, a lipodepsinonapeptide secreted by the majority of the strains of the bacterium, and a new form of syringopeptin, SP22Phv. The structure of the latter metabolite was elucidated by the combined use of mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy and chemical procedures. Comparative phytotoxic and antimicrobial assays showed that SP22Phv did not differ substantially from the previously characterized syringopeptin 22 (SP22) as far as toxicity to plants was concerned, but was less active in inhibiting the growth of the test fungi Rhodotorula pilimanae and Geotrichum candidum and of the Gram-positive bacterium Bacillus megaterium.