RESUMEN
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.
Asunto(s)
Bacterias/enzimología , Proteínas Bacterianas/química , Péptido Sintasas/química , Sintasas Poliquetidas/química , Fosfato de Piridoxal/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Secuencia Conservada , Evolución Molecular , Cadenas de Markov , Modelos Moleculares , Simulación del Acoplamiento Molecular , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Péptido Sintasas/metabolismo , Filogenia , Sintasas Poliquetidas/metabolismo , Conformación Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Péptidos Cíclicos/química , Fosfolípidos/química , Dodecil Sulfato de Sodio/química , Antifúngicos/química , Dicroismo Circular , Hemólisis , Enlace de Hidrógeno , Canales Iónicos/química , Lípidos/química , Micelas , Simulación de Dinámica Molecular , Conformación Proteica , Rifampin/farmacología , Agua/químicaRESUMEN
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.
Asunto(s)
Antifúngicos/farmacología , Proteínas Bacterianas/farmacología , Lipoproteínas/farmacología , Pseudomonas syringae/química , Rhizoctonia/efectos de los fármacos , Rhodotorula/efectos de los fármacos , Trichoderma/efectos de los fármacos , Aminoácidos/análisis , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Antifúngicos/química , Antifúngicos/aislamiento & purificación , Bacterias/efectos de los fármacos , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Eritrocitos/efectos de los fármacos , Hemólisis , Lipoproteínas/química , Lipoproteínas/aislamiento & purificación , Viabilidad Microbiana , Análisis EspectralRESUMEN
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.
Asunto(s)
Aminoaciltransferasas/química , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Modelos Moleculares , Péptido Sintasas/química , Pseudomonas syringae/metabolismo , Secuencia de Aminoácidos , Aminoaciltransferasas/genética , Aminoaciltransferasas/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Proteínas Portadoras/genética , Dimerización , Proteínas de Unión a Maltosa , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Oligopéptidos , Péptido Sintasas/genética , Péptido Sintasas/fisiología , Péptidos/genética , Conformación Proteica , Pseudomonas syringae/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Adenosina Trifosfato/química , Proteínas Bacterianas/química , Péptido Sintasas/química , Streptomyces/química , Treonina/análogos & derivados , Treonina/química , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Cinética , Modelos Moleculares , Péptido Sintasas/genética , Péptido Sintasas/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Streptomyces/enzimología , Especificidad por Sustrato , Treonina/biosíntesis , Treonina/metabolismoRESUMEN
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, SP(22)Phv. 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 SP(22)Phv did not differ substantially from the previously characterized syringopeptin 22 (SP(22)) 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.
Asunto(s)
Toxinas Bacterianas/biosíntesis , Fabaceae/microbiología , Péptidos Cíclicos/biosíntesis , Pseudomonas/metabolismo , Secuencia de Aminoácidos , Antibacterianos/biosíntesis , Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Antifúngicos/aislamiento & purificación , Antifúngicos/farmacología , Toxinas Bacterianas/aislamiento & purificación , Toxinas Bacterianas/farmacología , Cromatografía Líquida de Alta Presión , Espectroscopía de Resonancia Magnética/métodos , Espectrometría de Masas , Datos de Secuencia Molecular , Péptidos Cíclicos/aislamiento & purificación , Péptidos Cíclicos/farmacología , Pseudomonas/química , Especificidad de la Especie , Nicotiana/efectos de los fármacosRESUMEN
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: alpha-aminoadipate semialdehyde dehydrogenase, alpha-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.
Asunto(s)
Biología Computacional , Familia de Multigenes , Péptido Sintasas/química , Análisis de Secuencia de Proteína , Secuencia de Aminoácidos , Dominio Catalítico , Bases de Datos de Proteínas , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Pseudomonas syringae/enzimología , Streptomyces/enzimología , Treonina/análogos & derivados , Proteínas Bacterianas/genética , Biología Computacional , Datos de Secuencia Molecular , Familia de Multigenes/genética , Familia de Multigenes/fisiología , Mutagénesis Sitio-Dirigida , Relación Estructura-Actividad , Treonina/biosíntesisRESUMEN
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.