Project description:The ethylene-forming enzyme (EFE) from Pseudomonas syringae pv. phaseolicola PK2 is a member of the mononuclear non-heme Fe(II)- and 2-oxoglutarate (2OG)-dependent oxygenase superfamily. This enzyme is reported to simultaneously catalyze the conversion of 2OG into ethylene and three CO2 molecules and the Cδ hydroxylation of l-arginine (l-Arg) while oxidatively decarboxylating 2OG to form succinate and carbon dioxide. A new plasmid construct for expression in recombinant Escherichia coli cells allowed for the purification of large amounts of EFE with activity greater than that previously recorded. A variety of assays were used to quantify and confirm the identity of the proposed products, including the first experimental demonstration of l-Δ1-pyrroline-5-carboxylate and guanidine derived from 5-hydroxyarginine. Selected l-Arg derivatives could induce ethylene formation without undergoing hydroxylation, demonstrating that ethylene production and l-Arg hydroxylation activities are not linked. Similarly, EFE utilizes the alternative α-keto acid 2-oxoadipate as a cosubstrate (forming glutaric acid) during the hydroxylation of l-Arg, with this reaction unlinked from ethylene formation. Kinetic constants were determined for both ethylene formation and l-Arg hydroxylation reactions. Anaerobic UV-visible difference spectra were used to monitor the binding of Fe(II) and substrates to the enzyme. On the basis of our results and what is generally known about EFE and Fe(II)- and 2OG-dependent oxygenases, an updated model for the reaction mechanism is presented.
Project description:BackgroundEthylene is one of the most used chemical monomers derived from non-renewable sources and we are investigating the possibility of producing it in yeast via the ethylene forming enzyme (EFE) from Pseudomonas syringae. To enable engineering strategies to improve the enzyme, it is necessary to identify the regions and amino acid residues involved in ethylene formation.ResultsWe identified the open reading frame for the EFE homolog in Penicillium digitatum and also showed its capability of mediating ethylene production in yeast. The sequence of the EFE homologs from P.digitatum and P. syringae was compared to that of the non-functional EFE-homolog from Penicillium chrysogenum and ten amino acids were found to correlate with ethylene production. Several of these amino acid residues were found to be important for ethylene production via point mutations in P. syringae EFE. The EFE homolog from P. chrysogenum was engineered at 10 amino acid residues to mimic the P. syringae EFE, but this did not confer ethylene producing capability.Furthermore, we predicted the structure of EFE by homology to known structures of 2-oxoglutarate/Fe(II) dependent dioxygenases. Three of the amino acids correlating with ethylene production are located in the predicted 2-oxoglutarate binding domain. A protein domain specific for the EFE-class was shown to be essential for activity. Based on the structure and alanine substitutions, it is likely that amino acids (H189, D191 and H268) are responsible for binding the Fe(II) ligand.ConclusionWe provide further insight into the structure and function of the ethylene forming (EFE) - subclass of 2-oxoglutarate/Fe(II) dependent dioxygenases. We conclude that residues in addition to the 10 identified positions implicated in ethylene production by sequence comparison, are important for determining ethylene formation. We also demonstrate the use of an alternative EFE gene. The data from this study will provide the basis for directed protein engineering to enhance the ethylene production capability and properties of EFE.
Project description:Worldwide, ethylene is the most produced organic compound. It serves as a building block for a wide variety of plastics, textiles, and chemicals, and a process has been developed for its conversion into liquid transportation fuels. Currently, commercial ethylene production involves steam cracking of fossil fuels, and is the highest CO2-emitting process in the chemical industry. Therefore, there is great interest in developing technology for ethylene production from renewable resources including CO2 and biomass. Ethylene is produced naturally by plants and some microbes that live with plants. One of the metabolic pathways used by microbes is via an ethylene-forming enzyme (EFE), which uses ?-ketoglutarate and arginine as substrates. EFE is a promising biotechnology target because the expression of a single gene is sufficient for ethylene production in the absence of toxic intermediates. Here we present the first comprehensive review and analysis of EFE, including its discovery, sequence diversity, reaction mechanism, predicted involvement in diverse metabolic modes, heterologous expression, and requirements for harvesting of bioethylene. A number of knowledge gaps and factors that limit ethylene productivity are identified, as well as strategies that could guide future research directions.
Project description:The ethylene-forming enzyme (EFE) from Pseudomonas syringae pv. phaseolicola PK2 is a member of the mononuclear nonheme Fe(II)- and 2-oxoglutarate (2OG)-dependent oxygenase superfamily. EFE converts 2OG into ethylene plus three CO2 molecules while also catalyzing the C5 hydroxylation of l-arginine (l-Arg) driven by the oxidative decarboxylation of 2OG to form succinate and CO2. Here we report 11 X-ray crystal structures of EFE that provide insight into the mechanisms of these two reactions. Binding of 2OG in the absence of l-Arg resulted in predominantly monodentate metal coordination, distinct from the typical bidentate metal-binding species observed in other family members. Subsequent addition of l-Arg resulted in compression of the active site, a conformational change of the carboxylate side chain metal ligand to allow for hydrogen bonding with the substrate, and creation of a twisted peptide bond involving this carboxylate and the following tyrosine residue. A reconfiguration of 2OG achieves bidentate metal coordination. The dioxygen binding site is located on the metal face opposite to that facing l-Arg, thus requiring reorientation of the generated ferryl species to catalyze l-Arg hydroxylation. Notably, a phenylalanyl side chain pointing toward the metal may hinder such a ferryl flip and promote ethylene formation. Extensive site-directed mutagenesis studies supported the importance of this phenylalanine and confirmed the essential residues used for substrate binding and catalysis. The structural and functional characterization described here suggests that conversion of 2OG to ethylene, atypical among Fe(II)/2OG oxygenases, is facilitated by the binding of l-Arg which leads to an altered positioning of the carboxylate metal ligand, a resulting twisted peptide bond, and the off-line geometry for dioxygen coordination.
Project description:Pseudomonas savastanoi pv. savastanoi strains harbor native plasmids belonging to the pPT23A plasmid family (PFPs) which are detected in all pathovars of the related species Pseudomonas syringae examined and contribute to the ecological and pathogenic fitness of their host. However, there is a general lack of information about the gene content of P. savastanoi pv. savastanoi plasmids and their role in the interaction of this pathogen with olive plants. We designed a DNA macroarray containing 135 plasmid-borne P. syringae genes to conduct a global genetic analysis of 32 plasmids obtained from 10 P. savastanoi pv. savastanoi strains. Hybridization results revealed that the number of PFPs per strain varied from one to four. Additionally, most strains contained at least one plasmid (designated non-PFP) that did not hybridize to the repA gene of pPT23A. Only three PFPs contained genes involved in the biosynthesis of the virulence factor indole-3-acetic acid (iaaM, iaaH, and iaaL). In contrast, ptz, a gene involved in the biosynthesis of cytokinins, was found in five PFPs and one non-PFP. Genes encoding a type IV secretion system (T4SS), type IVA, were found in both PFPs and non-PFPs; however, type IVB genes were found only on PFPs. Nine plasmids encoded both T4SSs, whereas seven other plasmids carried none of these genes. Most PFPs and non-PFPs hybridized to at least one putative type III secretion system effector gene and to a variety of additional genes encoding known P. syringae virulence factors and one or more insertion sequence transposase genes. These results indicate that non-PFPs may contribute to the virulence and fitness of the P. savastanoi pv. savastanoi host. The overall gene content of P. savastanoi pv. savastanoi plasmids, with their repeated information, mosaic arrangement, and insertion sequences, suggests a possible role in adaptation to a changing environment.
Project description:The ethylene-forming enzyme (EFE), like many other 2-oxoglutarate (2OG)-dependent nonheme iron(II) oxygenases, catalyzes the oxidative decarboxylation of 2OG to succinate and CO2 to generate a highly reactive iron species that hydroxylates a specific alkane C-H bond, in this case targeting l-arginine (Arg) for hydroxylation. However, the prominently observed reactivity of EFE is the transformation of 2OG into ethylene and three molecules of CO2. Crystallographic and biochemical studies have led to several proposed mechanisms for this 2-fold reactivity, but the detailed reaction steps are still obscure. Here, the thermodynamics associated with iron(II), 2OG, and Arg binding to EFE are studied using calorimetry (isothermal titration calorimetry and differential scanning calorimetry) to gain insight into how these binding equilibria organize the active site of EFE, which may have an impact on the O2 activation pathways observed in this system. Calorimetric data show that the addition of iron(II), Arg, and 2OG increases the stability over that of the apoenzyme, and there is distinctive cooperativity between substrate and cofactor binding. The energetics of binding of 2OG to Fe·EFE are consistent with a unique monodentate binding mode, which is different than the prototypical 2OG coordination mode in other 2OG-dependent oxygenases. This difference in the pre-O2 activation equilibria may be important for supporting the alternative ethylene-forming chemistry of EFE.
Project description:The Pseudomonas savastanoi species comprises a group of phytopathogenic bacteria that cause symptoms of disease in woody hosts. This is mediated by the rapid activation of a pool of virulence factors that suppress host defences and hijack the host's metabolism to the pathogen's benefit. The hrpL gene encodes an essential transcriptional regulator of virulence functions, including the type III secretion system (T3SS), in pathogenic bacteria. Here, we analyzed the contribution of HrpL to the virulence of four pathovars (pv.) of P. savastanoi isolated from different woody hosts (oleander, ash, broom, and dipladenia) and characterized the HrpL regulon of P. savastanoi pv. savastanoi NCPPB 3335 using two approaches: whole transcriptome sequencing (RNA-seq) and the bioinformatic prediction of candidate genes containing an hrp-box. Pathogenicity tests carried out for the P. savastanoi pvs. showed that HrpL was essential for symptom development in both non-host and host plants. The RNA-seq analysis of the HrpL regulon in P. savastanoi revealed a total of 53 deregulated genes, 49 of which were downregulated in the ΔhrpL mutant. Bioinformatic prediction resulted in the identification of 50 putative genes containing an hrp-box, 16 of which were shared with genes previously identified by RNA-seq. Although most of the genes regulated by HrpL belonged to the T3SS, we also identified some genes regulated by HrpL that could encode potential virulence factors in P. savastanoi.
Project description:GacS/GacA is a widely distributed two-component system playing an essential role as a key global regulator, although its characterization in phytopathogenic bacteria has been deeply biased, being intensively studied in pathogens of herbaceous plants but barely investigated in pathogens of woody hosts. P. savastanoi pv. savastanoi (Psv) is characterized by inducing tumours in the stem and branches of olive trees. In this work, the model strain Psv NCPPB 3335 and a mutant derivative with a complete deletion of gene gacA were subjected to RNA-Seq analyses in a minimum medium and a medium mimicking in planta conditions, accompanied by RT-qPCR analyses of selected genes and phenotypic assays. These experiments indicated that GacA participates in the regulation of at least 2152 genes in strain NCPPB 3335, representing 37.9 % of the annotated CDSs. GacA also controls the expression of diverse rsm genes, and modulates diverse phenotypes, including motility and resistance to oxidative stresses. As occurs with other P. syringae pathovars of herbaceous plants, GacA regulates the expression of the type III secretion system and cognate effectors. In addition, GacA also regulates the expression of WHOP genes, specifically encoded in P. syringe strains isolated from woody hosts, and genes for the biosynthesis of phytohormones. A gacA mutant of NCPPB 3335 showed increased virulence, producing large immature tumours with high bacterial populations, but showed a significantly reduced competitiveness in planta. Our results further extend the role of the global regulator GacA in the virulence and fitness of a P. syringae pathogen of woody hosts.
Project description:Erwinia toletana was first reported in 2004 as a bacterial species isolated from olive knots caused by the plant bacterium Pseudomonas savastanoi pv. savastanoi. Recent studies have shown that the presence of this bacterium in the olive knot environment increases the virulence of the disease, indicating possible interspecies interactions with P. savastanoi pv. savastanoi. Here, we report the first draft genome sequence of an E. toletana strain.