Phaseolicola PK2 Research Articles

Structures and Mechanisms of the Non-Heme Fe(II)- and 2-Oxoglutarate-Dependent Ethylene-Forming Enzyme: Substrate Binding Creates a Twist.

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.

Correction to Biochemical and Spectroscopic Characterization of the Non-Heme Fe(II)- and 2-Oxoglutarate-Dependent Ethylene-Forming Enzyme from Pseudomonas syringae pv. phaseolicola PK2.

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 ...

Ethylene production with engineered Synechocystis sp PCC 6803 strains

BackgroundMetabolic engineering and synthetic biology of cyanobacteria offer a promising sustainable alternative approach for fossil-based ethylene production, by using sunlight via oxygenic photosynthesis, to convert carbon dioxide directly into ethylene. Towards this, both well-studied cyanobacteria, i.e., Synechocystis sp PCC 6803 and Synechococcus elongatus PCC 7942, have been engineered to produce ethylene by introducing the ethylene-forming enzyme (Efe) from Pseudomonas syringae pv. phaseolicola PK2 (the Kudzu strain), which catalyzes the conversion of the ubiquitous tricarboxylic acid cycle intermediate 2-oxoglutarate into ethylene.ResultsThis study focuses on Synechocystis sp PCC 6803 and shows stable ethylene production through the integration of a codon-optimized version of the efe gene under control of the Ptrc promoter and the core Shine–Dalgarno sequence (5′-AGGAGG-3′) as the ribosome-binding site (RBS), at the slr0168 neutral site. We have increased ethylene production twofold by RBS screening and further investigated improving ethylene production from a single gene copy of efe, using multiple tandem promoters and by putting our best construct on an RSF1010-based broad-host-self-replicating plasmid, which has a higher copy number than the genome. Moreover, to raise the intracellular amounts of the key Efe substrate, 2-oxoglutarate, from which ethylene is formed, we constructed a glycogen-synthesis knockout mutant (ΔglgC) and introduced the ethylene biosynthetic pathway in it. Under nitrogen limiting conditions, the glycogen knockout strain has increased intracellular 2-oxoglutarate levels; however, surprisingly, ethylene production was lower in this strain than in the wild-type background.ConclusionMaking use of different RBS sequences, production of ethylene ranging over a 20-fold difference has been achieved. However, a further increase of production through multiple tandem promoters and a broad-host plasmid was not achieved speculating that the transcription strength and the gene copy number are not the limiting factors in our system.

Biochemical and Spectroscopic Characterization of the Non-Heme Fe(II)- and 2-Oxoglutarate-Dependent Ethylene-Forming Enzyme from Pseudomonas syringae pv. phaseolicola PK2.

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.

Ethylene Formation and Phenotypic Analysis of Transgenic Tobacco Plants Expressing a Bacterial Ethylene-Forming Enzyme

A bacterial ethylene-forming enzyme (EFE) catalyzes oxygenation of 2-oxoglutarate to produce ethylene and carbon dioxide in contrast to a plant enzyme which uses 1-aminocyclopropane-1-carboxylic acid as a substrate. We constructed several lines of transgenic tobacco plants which expressed an EFE from Pseudomonas syringae pv. phaseolicola PK2. The gene encoding a chimeric protein consisting of EFE and beta-glucuronidase (GUS) was introduced into the tobacco genome using a binary vector which directs expression of the EFE-GUS fusion protein under the control of constitutive promoter of cauliflower mosaic virus 35S RNA. Two lines of transgenic plants produced ethylene at consistently higher rates than the untransformed plant, and their GUS activities were expressed in different tissues. A significant dwarf morphology observed in the transgenic tobacco displaying the highest ethylene production resembled the phenotype of a wild-type plant exposed to excess ethylene. These results demonstrate a potential use of bacterial EFE to supply ethylene as a hormonal signal via an alternative route using an ubiquitous substrate 2-oxoglutarate in plant tissues.

Detection of New Ethylene-Producing Bacteria, Pseudomonas syringae pvs. cannabina and sesami, by PCR Amplification of Genes for the Ethylene-Forming Enzyme

ABSTRACT Strains of Pseudomonas syringae (78 strains and 43 pathovars) and other strains (79) of plant and insect origin were examined for the presence of the ethylene-forming enzyme gene (efe) by polymerase chain reaction (PCR) assay. The sequence of the efe gene of P. syringae pv. phaseolicola PK2 was used to design two primer sets for amplification of the gene. In addition to P.syringae pv. phaseolicola (the "kudzu strain") and P.syringae pv. glycinea, which were efficient ethylene producers, several strains of P.syringae pvs. sesami and cannabina generated PCR products of the predicted size. A DNA probe of the efe gene, isolated from strain PK2, hybridized to these PCR products, indicating homology to the P.syringae pv. phaseolicola efe gene. PCR restriction fragment length polymorphism analyses suggested that these four pathovars harbor a similar efe gene. Furthermore, the probe hybridized to an indigenous plasmid of P.syringae pv. cannabina, suggesting that the efe gene could be located on a plasmid in this pathovar, but did not hybridize to plas-mids of P.syringae pv. sesami strains. P.syringae pvs. sesami and cannabina strains produced ethylene in King's medium B at levels similar to those of P.syringae pvs. phaseolicola and glycinea. Thus, two new ethylene-producing bacteria were detected by the PCR assay.

Photosynthetic conversion of carbon dioxide to ethylene by the recombinant cyanobacterium, Synechococcus sp. PCC 7942, which harbors a gene for the ethylene-forming enzyme of Pseudomonas syringae

Photosynthetic conversion of carbon dioxide to ethylene was studied using the recombinant cyanobacterium, Synechococcus sp. strain PCC 7942 R2-SPc which expresses the ethylene-forming enzyme (EFE) from Pseudomonas syringae pv. phaseolicola PK2. The gene encoding the EFE ( efe gene) from P. syringae was introduced into the cyanobacterium utilizing the pUC303 shuttle vector into which the efe gene was placed under the control of various transcriptional signals, i.e., the native promoter and terminator of the efe gene (pUC303-EFE03), the Escherichia coli lacZ promoter and the efe gene terminator (pUC303-EFE10 and pUC303-EFE30), or the promoter and terminator of the psbAI gene from Synechococcus sp. PCC7942 which codes for the D1 protein in photosystem II (pEXE3). Among these configurations, EFE activity measured in the cell-free extracts of transformants that harbored the pEXE3 was highest. However, ethylene production in vivo of the transformants carrying pEXE3 declined with the number of generations, because homologous recombination of DNA sequences on the pEXE3 plasmid and host chromosomal psbAI locus took place. Deletion of the 5′-upstream region of the psbAI promoter and the 3′-downstream region of the psbAI terminator in pEXE3 resulted in pEXE3Δ8 which showed the highest level of ethylene-forming activity, although the latter plasmid was still unstable with a half-life of only 12 generations. The amount of carbon incorporated into ethylene was calculated as a percentage of the total carbon fixed, the maximum value of which was 5.84% in the recombinant cyanobacterium harboring pUC303-EFE03.

Ethylene Production by Pseudomonas syringae Pathovars In Vitro and In Planta

Significant amounts of ethylene were produced by Pseudomonas syringae pv. glycinea, pv. phaseolicola (which had been isolated from viny weed Pueraria lobata [Willd.] Ohwi [common name, kudzu]), and pv. pisi in synthetic medium. On the other hand, the bean strains of P. syringae pv. phaseolicola and strains of 17 other pathovars did not produce ethylene. P. syringae pv. glycinea and P. syringae pv. phaseolicola produced nearly identical levels of ethylene (about 5 x 10(sup-7) nl h(sup-1) cell(sup-1)), which were about 10 times higher than the ethylene level of P. syringae pv. pisi. Two 22-bp oligonucleotide primers derived from the ethylene-forming enzyme (efe) gene of P. syringae pv. phaseolicola PK2 were investigated for their ability to detect ethylene-producing P. syringae strains by PCR analysis. PCR amplification with this primer set resulted in a specific 0.99-kb fragment in all ethylene-producing strains with the exception of the P. syringae pv. pisi strains. Therefore, P. syringae pv. pisi may use a different biosynthetic pathway for ethylene production or the sequence of the efe gene is less conserved in this bacterium. P. syringae pv. phaseolicola isolated from kudzu and P. syringae pv. glycinea also produced ethylene in planta. It could be shown that the enhanced ethylene production in diseased tissue was due to the production of ethylene by the inoculated bacteria. Ethylene production in vitro and in planta was strictly growth associated.

Ethylene production using a broad-host-range plasmid in Pseudomonas syringae and Pseudomonas putida

We have constructed a high-copy-number, broad-host-range plasmid, pMEFE1 that carries a gene encoding an ethylene-forming enzyme from Pseudomonas syringae pv. phaseolicola PK2. The ethylene-forming enzyme was expressed under the control of a promoter of the neomycin phosphotransferase gene on pMEFE1. When introduced into P. syringae and Pseudomonas putida cells by transconjugation, pMEFE1 conferred on the recombinant cells the ability to synthesize the ethylene-forming enzyme with specific activities 41 and 20 times higher, respectively, than that of the ethylene-forming enzyme in the parental P. syringae.

Overexpression and in vitro reconstitution of the ethylene-forming enzyme from Pseudomonas syringae

By replacing a native promoter with lac and tac promoters, the gene encoding an ethylene-forming enzyme (EFE) from Pseudomonas syringae pv. phaseolicola PK2 was overexpressed in Escherichia coli. The EFE protein expressed by a multicopy plasmid accounted for more than 30% of the total cellular protein, resulting in ethylene-forming activities higher than 10 μl of ethylene (mg cell) −1h −1 in recombinant E. coli cells. However, most of the EFE protein accumulated as inactive inclusion bodies particularly at elevated temperatures (>30°C). We present an efficient procedure for reconstituting an active enzyme from inclusion bodies by solubilization with 8 M urea and dialysis. The reconstituted EFE has specific activity identical to that of the native enzyme from P. syringae, suggesting that the EFE protein has an intrinsic folding capability in vitro.

Ethylene production by strains of the plant-pathogenic bacterium Pseudomonas syringae depends upon the presence of indigenous plasmids carrying homologous genes for the ethylene-forming enzyme

The molecular characteristics of the ethylene-forming enzymes of strains of Pseudomonas syringae were tested. The ethylene-producing activities of the nine strains as measured in vivo and in vitro were similar, except for that of P. syringae pv. mori M5. A polyclonal antibody and a DNA probe for the ethylene-forming enzyme from P. syringae pv. phaseolicola PK2 were prepared to investigate homologies among the proteins and genes for the ethylene-forming enzymes. With the exception of P. syringae pv. mori M5, eight strains tested expressed the same antigen as the ethylene-forming enzyme from P. syringae pv. phaseolicola PK2 and were homologous to DNA sequences on indigenous plasmids. Molecular masses of antigenic proteins from all ethylene-producing strains were 40 kDa. The N-terminal amino acid sequence of the purified ethylene-forming enzyme from P. syringae pv. glycinea KN130 was identical to that of the enzyme from P. syringae pv. phaseolicola PK2. These results show that the ethylene-forming enzymes encoded by the indigenous plasmid(s) in the pathogenic bacteria examined were similar.

Molecular cloning in Escherichia coli, expression, and nucleotide sequence of the gene for the ethylene-forming enzyme of Pseudomonas syringae pv. phaseolicola PK2

The gene for the ethylene-forming enzyme of Pseudomonas syringae pv. phaseolicola PK2 was found to be encoded by an indigenous plasmid, designated pPSP1. The gene for the ethylene-forming enzyme was cloned and expressed in Escherichia coli JM109. Nucleotide sequence analysis of the clone revealed an open reading frame that encodes 350 amino acids (mol. wt. 39,444). In a comparison with other proteins, the homology score for the entire amino-acid sequence of the ethylene-forming enzyme of Pseudomonas syringae versus ethylene-forming enzymes from plants and 2-oxoglutarate-dependent dioxygenases was low. However, functionally significant regions are conserved.

Two reactions are simultaneously catalyzed by a single enzyme: The arginine-dependent simultaneous formation of two products, ethylene and succinate, from 2-oxoglutarate by an enzyme from Pseudomonas syringae

A single enzyme isolated from Pseudomonas syringae pv. phaseolicola PK2 simultaneously catalyzed two reactions, namely, the formation of ethylene and succinate from 2-oxoglutarate, at a molar ratio of 2:1. In the main reaction, 2-oxoglutarate was dioxygenated to produce one molecule of ethylene and three molecules of carbon dioxide. In the sub-reaction, both 2-oxoglutarate and L-arginine were mono-oxygenated to yield succinate plus carbon dioxide and L-hydroxyarginine, respectively, the latter being further transformed to guanidine and L- δ 1-pyrroline-5-carboxylate. We propose a dual-circuit mechanism for the entire reaction, in which the binding of L-arginine and 2-oxoglutarate in a Schiff-base structure generates a common intermediate for two reactions.

Classification of ethylene-producing bacteria in terms of biosynthetic pathways to ethylene

Abstract Two hundred and twenty-nine ethylene-producing strains of bacteria were identified among 757 bacterial strains which included 13 strains of chemolithotrophs. The ethylene-producing bacetria were classified into three groups, namely, l -methionine-dependent, 2-ketoglutarate-dependent and meat extract-dependent, with reference to their respective biosynthetic pathways to ethylene. Two hundred and twenty-five l -methionine-dependent strains were obtained, while the only 2-ketoglutarate-dependent strain was Pseudomonas syringae pv. phaseolicola PK2. Three strains of chemolithotrophs had ethylene-forming capacity, and Thiobacillus novellus IFO 12443 had a novel ethylene-forming system which was dependent upon the addition of meat extract into the culture medium. The ethylene-forming systems of two of the strains of Thiobacillus ferrooxidans have not yet been characterized. Several strains of non-ethylene-producing bacteria failed to produce ethylene, even when l -methionine was added to the culture medium. We examined the causes of their lack of ethylene-producing ability and judged that these strains are either NADH:Fe(III)EDTA oxidoreductase-less or methionine-uptake activity-less.

Purification and properties of an ethylene-forming enzyme from Pseudomonas syringae pv. phaseolicola PK2

A novel ethylene-forming enzyme that catalyses the formation of ethylene from 2-oxoglutarate was purified from a cell-free extract of Pseudomonas syringae pv. phaseolicola PK2. It was purified about 2800-fold with an overall yield of 53% to a single band of protein after SDS-PAGE. The purified enzyme had a specific activity of 660 nmol ethylene min-1 (mg protein)-1. The molecular mass of the enzyme was approximately 36 kDa by gel filtration and 42 kDa by SDS-PAGE. The isoelectric point and optimum pH were 5.9 and ca. 7.0-7.5, respectively. There was no homology between the N-terminal amino acid sequence of the ethylene-forming enzyme of Ps. syringae pv. phaseolicola PK2 and the sequence of the ethylene-forming enzyme of the fungus Penicillium digitatum IFO 9372. However, the two enzymes have the following properties in common. The presence of 2-oxoglutarate, L-arginine, Fe2+ and oxygen is essential for the enzymic reaction. The enzymes are highly specific for 2-oxoglutarate as substrate and L-arginine as cofactor. EDTA, Tiron, DTNB [5,5'-dithio-bis(2-nitrobenzoate)] and hydrogen peroxide are all effective inhibitors.

L-Arginine is essential for the formation in vitro of ethylene by an extract of Pseudomonas syringae

Summary: A system was developed for the formation of ethylene in vitro by an extract of Pseudomonas syringae pv. phaseolicola PK2. The ethylene-forming activity of a cell-free extract of this bacterium measured in a system reported previously was almost completely lost when the cell-free extract was dialysed against potassium phosphate buffer for 24 h at 4 °C. When the fraction of cell-free extract with a molecular mass < 10 kDa (SupI) was added back to the enzyme fraction after gel filtration of the cell-free extract, the enzymic activity increased to about four times that of the gel-filtered crude enzyme. The action of SupI could be reproduced by the addition of l-arginine. The complete system for the formation of ethylene under aerobic conditions in vitro required 0·25 mm-2-oxoglutarate, 0·2 mm-FeSO4, 2 mm-DTT, 10 mm-l-histidine and 0·2 mm-l-arginine. The cofactor specificity was examined by replacing l-arginine or l-histidine with various analogues, but none of them were effective. The components of this system, with the exception of l-histidine, were similar to those of a system derived from the ethylene-producing, plant pathogenic fungus Penicillium digitatum which also produced ethylene in vitro in a reaction dependent on 2-oxoglutarate. The intermediates in the ethylene-forming reaction are postulated and the roles of l-arginine and l-histidine in the formation of ethylene by Ps. syringae are discussed.