Yuri Gogolev

Tomato CYP74C3 is a Multifunctional Enzyme not only Synthesizing Allene Oxide but also Catalyzing its Hydrolysis and Cyclization

The mechanism of the recombinant tomato allene oxide synthase (LeAOS3, CYP74C3) was studied. Incubations of linoleic acid (9S)‐hydroperoxide with dilute suspensions of LeAOS3 (10–20 s, 0 °C) yield mostly the expected allene oxide (12Z)‐9,10‐epoxy‐10,12‐octadecadienoic acid (9,10‐EOD), which was detected as its methanol‐trapping product. In contrast, the relative yield of 9,10‐EOD progressively decreased when the incubations were performed with fourfold, tenfold, or 80‐fold larger amounts of LeAOS3, while α‐ketol and the cyclopentenone rac‐cis‐10‐oxo‐11‐phytoenoic acid (10‐oxo‐PEA) became the predominant products. Both the α‐ketol and 10‐oxo‐PEA were also produced when LeAOS3 was exposed to preformed 9,10‐EOD, which was generated by maize allene oxide synthase (CYP74A). LeAOS3 also converted linoleic acid (13S)‐hydroperoxide into the corresponding allene oxide, but with about tenfold lower yield of cyclopentenone. The results indicate that in contrast to the ordinary allene oxide synthases (CYP74A subfamily), LeAOS3 (CYP74C subfamily) is a multifunctional enzyme, catalyzing not only the synthesis, but also the hydrolysis and cyclization of allene oxide.

Isolation and characterization of a glyphosate-degrading rhizosphere strain, Enterobacter cloacae K7.

Plant-growth-promoting rhizobacteria exert beneficial effects on plants through their capacity for nitrogen fixation, phytohormone production, phosphate solubilization, and improvement of the water and mineral status of plants. We suggested that these bacteria may also have the potential to express degradative activity toward glyphosate, a commonly used organophosphorus herbicide. In this study, 10 strains resistant to a 10 mM concentration of glyphosate were isolated from the rhizoplane of various plants. Five of these strains--Alcaligenes sp. K1, Comamonas sp. K4, Azomonas sp. K5, Pseudomonas sp. K3, and Enterobacter cloacae K7--possessed a number of associative traits, including fixation of atmospheric nitrogen, solubilization of phosphates, and synthesis of the phytohormone indole-3-acetic acid. One strain, E. cloacae K7, could utilize glyphosate as a source of P. Gas-liquid chromatography showed that E. cloacae growth correlated with a decline in herbicide content in the culture medium (40% of the initial 5mM content), with no glyphosate accumulating inside the cells. Thin-layer chromatography analysis of the intermediate metabolites of glyphosate degradation found that E. cloacae K7 had a C-P lyase activity and degraded glyphosate to give sarcosine, which was then oxidized to glycine. In addition, strain K7 colonized the roots of common sunflower (Helianthus annuus L.) and sugar sorghum (Sorghum saccharatum Pers.), promoting the growth and development of sunflower seedlings. Our findings extend current knowledge of glyphosate-degrading rhizosphere bacteria and may be useful for developing a biotechnology for the cleanup and restoration of glyphosate-polluted soils.

Supramolecular Systems Based on Novel Mono- and Dicationic Pyrimidinic Amphiphiles and Oligonucleotides: A Self-Organization and Complexation Study

Novel mono- and dicationic pyrimidinic surfactants are synthesized and their aggregation behavior is studied by methods of tensiometry and nuclear magnetic resonance (NMR) self-diffusion. To estimate their potentiality as gene delivery agents, the complexation with oligonucleotides (ONus) is explored by dynamic light scattering (DLS) and zeta-potential titration methods and ethidium bromide exclusion experiments. Bola-type pyrimidinic amphiphile (BPM) demonstrates rather a weak affinity to ONus. Although it induces mixed associations with ONus, only slight charge compensation changes occur at a large excess of bola, with no recharging reached. Similarly, the ethydium bromide exclusion study reveals a slow increase in the binding capacity toward an ONu with an increment in BPM concentration. The monocationic pyrimidinic surfactant (MPM) and its gemini analogue (GPM-1) are ranked as intermediates in both their aggregative activity and complexing properties toward ONus. They both form mixed associates with ONus well below the critical micelle concentrations (cmcs) of 2 and 15 mM respectively. However, GPM-1 has a much lower isoelectric point at the molar ratio surfactant/ONu r~1 compared to r~3 for MPM. This probably indicates a larger electrostatic contribution to the ONu complexation in the case of GPM-1. The most hydrophobic pyrimidinic surfactant (GPM-2), bearing three alkyl tails, demonstrates enhanced aggregative activity and binding capacity toward ONus as compared to former pyrimidinic surfactants. Due to effective aggregative (low cmc of 0.04 mM) plus binding properties (fraction of bound ONu β=0.76 at r=2.5), GPM-2 may be ranked as a promising agent for wider biological applications.

Determinants governing the CYP74 catalysis: Conversion of allene oxide synthase into hydroperoxide lyase by site-directed mutagenesis

Bioinformatics analyses enabled us to identify the hypothetical determinants of catalysis by CYP74 family enzymes. To examine their recognition, two mutant forms F295I and S297A of tomato allene oxide synthase LeAOS3 (CYP74C3) were prepared by site‐directed mutagenesis. Both mutations dramatically altered the enzyme catalysis. Both mutant forms possessed the activity of hydroperoxide lyase, while the allene oxide synthase activity was either not detectable (F295I) or significantly reduced (S297A) compared to the wild‐type LeAOS3. Thus, both sites 295 and 297 localized within the “I‐helix central domain” (“oxygen binding domain”) are the primary determinants of CYP74 type of catalysis.

Hydroperoxide lyase cascade in pea seedlings: Non-volatile oxylipins and their age and stress dependent alterations.

The profiles of non-volatile oxylipins of pea (Pisum sativum) seedlings were examined by gas chromatography-mass spectrometry after invitro incubation with α-linolenic acid. The 13-lipoxygenase/hydroperoxide lyase (HPL) products were predominant in the leaves, while the roots possess both 13- and 9-HPL products. Allene oxide synthase (AOS) and divinyl ether synthase (DES) products were not detected in the leaves or in the roots of any age. The HPL cascade produces a diversity of oxylipins, including the compounds (2E)-4-hydroxy-traumatic, (10E)-9,12-dihydroxy-10-dodecenoic and 9,12-dihydroxydodecanoic acids, as well as (2E)-4-hydroxy-2-nonenoic acid, which has not yet been detected in plants. Oxylipin patterns were altered by infection, water deficit, as well as by plant age. Infection caused the specific strong accumulation of azelaic (nonane-1,9-dioic) acid in the leaves. The azelaic acid content in the aged (14 and 18day-old) leaves was significantly higher than in the younger leaves. Water deficit induced the accumulation of (2E)-4-hydroxy-2-nonenoic acid and (2E)-traumatic acid in the roots. Results demonstrate that: (1) the HPL cascade is the predominant branch of the lipoxygenase pathway in pea seedlings; (2) the HPL products may have the regulatory role both in growth control and adaptation.

Structure–function relationship in the CYP74 family: Conversion of divinyl ether synthases into allene oxide synthases by site-directed mutagenesis

Non‐classical P450s of CYP74 family control several enzymatic conversions of fatty acid hydroperoxides to bioactive oxylipins in plants, some invertebrates and bacteria. The family includes two dehydrases, namely allene oxide synthase (AOS) and divinyl ether synthase (DES), and two isomerases, hydroperoxide lyase (HPL) and epoxyalcohol synthase. To study the interconversion of different CYP74 enzymes, we prepared the mutant forms V379F and E292G of tobacco (CYP74D3) and flax (CYP74B16) divinyl ether synthases (DESs), respectively. In contrast to the wild type (WT) enzymes, both mutant forms lacked DES activity. Instead, they produced the typical AOS products, α‐ketols and (in the case of the flax DES mutant) 12‐oxo‐10,15‐phytodienoic acid. This is the first demonstration of DES into AOS conversions caused by single point mutations.

Cell-to-cell communication in the populations of enterobacterium Erwinia carotovora ssp. atroseptica SCRI1043 during adaptation to stress conditions

Erwinia carotovora ssp. atroseptica SCRI1043 is a plant pathogenic bacterium. Many species of enterobacteria including Erwinia sp. can induce the onset or the progression of opportunistic or persistent infections in humans. The existence of these bacteria within different ecological niches is related to their significant adaptive potential. The triggering of adaptive reactions, which are needed for bacterial persistence, is controlled in many cases by intercellular communication; hence, the ability to survive under unfavourable conditions is regulated in a cell-density-dependent manner. In this study, we showed that, during starvation of E. carotovora ssp. atroseptica SCRI1043, the initial stage of the response to stress was stabilization of the density of culturable cells in the range 10(6)-10(7) CFU mL(-1). The number of culturable cells increased (up to approximately 10(6) CFU mL(-1)) when cultures were inoculated at a low cell density (10(3)-10(5) CFU mL(-1)), and at the same time, acylhomoserine lactone (AHL)-dependent quorum sensing was activated. Our results showed that the regulation of cell density in starving populations of E. carotovora ssp. atroseptica SCRI1043 occurred and this regulation was carried out with the involvement of the cell-to-cell communication.

Transcriptome portrait of cellulose-enriched flax fibres at advanced stage of specialization

Functional specialization of cells is among the most fundamental processes of higher organism ontogenesis. The major obstacle to studying this phenomenon in plants is the difficulty of isolating certain types of cells at defined stages of in planta development for in-depth analysis. A rare opportunity is given by the developed model system of flax (Linum usitatissimum L.) phloem fibres that can be purified from the surrounding tissues at the stage of the tertiary cell wall deposition. The performed comparison of the whole transcriptome profile in isolated fibres and other portions of the flax stem, together with fibre metabolism characterization, helped to elucidate the general picture of the advanced stage of plant cell specialization and to reveal novel participants potentially involved in fibre metabolism regulation and cell wall formation. Down-regulation of all genes encoding proteins involved in xylan and lignin synthesis and up-regulation of genes for the specific set of transcription factors transcribed during tertiary cell wall formation were revealed. The increased abundance of transcripts for several glycosyltransferases indicated the enzymes that may be involved in synthesis of fibre-specific version of rhamnogalacturonan I.

Genome Sequence of Pectobacterium atrosepticum Strain 21A

ABSTRACT We report the annotated genome sequence of the enterobacterial plant pathogen Pectobacterium atrosepticum strain 21A, isolated in Belarus from potato stem with blackleg symptoms.

Specificity of oxidation of linoleic acid homologs by plant lipoxygenases

The lipoxygenase-catalyzed oxidation of linoleic acid homologs was studied. While the linoleic acid oxidation by maize 9-lipoxygenase (9-LO) specifically produced (9S)-hydroperoxide, the dioxygenation of (11Z,14Z)-eicosadienoic (20:2) and (13Z,16Z)-docosadienoic (22:2) acids by the same enzyme lacked regio- and stereospecificity. The oxidation of 20:2 and 22:2 by 9-LO afforded low yields of racemic 11-, 12-, 14-, and 15-hydroperoxides or 13- and 17-hydroperoxides, respectively. Soybean 13-lipoxygenase-1 (13-LO) specifically oxidized 20:2, 22:2, and linoleate into (ω6S)-hydroperoxides. Dioxygenation of (9Z,12Z)-hexadecadienoic acid (16:2) by both 9-LO and 13-LO occurred specifically, affording (9S)- and (13S)-hydroperoxides, respectively. The data are consistent with the “pocket theory of lipoxygenase catalysis” (i.e. with the penetration of a substrate into the active center with the methyl end first). Our findings also demonstrate that the distance between carboxyl group and double bonds substantially determines the positioning of substrates within the active site.