E. V. Osipova

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.

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.

Structure of Scots pine defensin 1 by spectroscopic methods and computational modeling.

Defensins are part of the innate immune system in plants with activity against a broad range of pathogens, including bacteria, fungi and viruses. Several defensins from conifers, including Scots pine defensin 1 (Pinus sylvestris defensin 1, (PsDef1)) have shown a strong antifungal activity, however structural and physico-chemical properties of the family, needed for establishing the structure-dynamics-function relationships, remain poorly characterized. We use several spectroscopic and computational methods to characterize the structure, dynamics, and oligomeric state of PsDef1. The three-dimensional structure was modeled by comparative modeling using several programs (Geno3D, SWISS-MODEL, I-TASSER, Phyre(2), and FUGUE) and verified by circular dichroism (CD) and infrared (FTIR) spectroscopy. Furthermore, FTIR data indicates that the structure of PsDef1 is highly resistant to high temperatures. NMR diffusion experiments show that defensin exists in solution in the equilibrium between monomers and dimers. Four types of dimers were constructed using the HADDOCK program and compared to the known dimer structures of other plant defensins. Gaussian network model was used to characterize the internal dynamics of PsDef1 in monomer and dimer states. PsDef1 is a typical representative of P. sylvestris defensins and hence the results of this study are applicable to other members of the family.

Alteration of catalysis of CYP74C subfamily enzymes as a result of site-directed mutagenesis

287 Enzymes of the lipoxygenase signalling system— lipoxygenases and cytochromes P450 of the CYP74 family, which catalyze the formation of oxylipins, oxi dizing derivatives of fatty acids—play a key role in plant development and response to stressors [1–4]. There are three known kinds of CYP74 enzymes: two dehydrases, namely allene oxide synthase (AOS) and divinyl ether synthase (DES) and one isomerase, called hydroperoxide lyase (HPL).

Hexadecanoid pathway in plants: Lipoxygenase dioxygenation of (7Z,10Z,13Z)-hexadecatrienoic acid

Abstract7,10,13-Hexadecatrienoic acid (16:3) is abundant in many plant species. However, its metabolism through the lipoxygenase pathway is not sufficiently understood. The goal of present work was to investigate the oxygenation of 16:3 by different plant lipoxygenases and to study the occurrence of oxygenated derivatives of 16:3 in plant seedlings. The recombinant maize 9-lipoxygenase specifically converted 16:3 into (7S)-hydroperoxide. Identification of this novel oxylipin was substantiated by data of GC-MS, LC-MS/MS, 1H-NMR, and 2D-COSY as well as by deuterium labeling from [2H6]16:3. Soybean lipoxygenase 1 produced 91% (11S)-hydroperoxide and 6% racemic 14-hydroperoxide. Recombinant soybean lipoxygenase 2 (specifically oxidizing linoleate into 13-hydroperoxide) lacked any specificity towards 16:3. Lipoxygenase 2 produced 7-, 8-, 10-, 11-, 13-, and 14-hydroperoxides of 16:3, as well as a significant amount of bis-allylic 9-hydroperoxide. Seedlings of several examined plant species possessed free hydroxy derivatives of 16:3 (HHTs), as well as their ethyl esters. Interestingly, HHTs occur not only in “16:3 plants”, but also in typical “18:3 plants” like pea and soybean seedlings.

Global Gene Expression Analysis of Cross-Protected Phenotype of Pectobacterium atrosepticum.

The ability to adapt to adverse conditions permits many bacterial species to be virtually ubiquitous and survive in a variety of ecological niches. This ability is of particular importance for many plant pathogenic bacteria that should be able to exist, except for their host plants, in different environments e.g. soil, water, insect-vectors etc. Under some of these conditions, bacteria encounter absence of nutrients and persist, acquiring new properties related to resistance to a variety of stress factors (cross-protection). Although many studies describe the phenomenon of cross-protection and several regulatory components that induce the formation of resistant cells were elucidated, the global comparison of the physiology of cross-protected phenotype and growing cells has not been performed. In our study, we took advantage of RNA-Seq technology to gain better insights into the physiology of cross-protected cells on the example of a harmful phytopathogen, Pectobacterium atrosepticum (Pba) that causes crop losses all over the world. The success of this bacterium in plant colonization is related to both its virulence potential and ability to persist effectively under various stress conditions (including nutrient deprivation) retaining the ability to infect plants afterwards. In our previous studies, we showed Pba to be advanced in applying different adaptive strategies that led to manifestation of cell resistance to multiple stress factors. In the present study, we determined the period necessary for the formation of cross-protected Pba phenotype under starvation conditions, and compare the transcriptome profiles of non-adapted growing cells and of adapted cells after the cross-protective effect has reached the maximal level. The obtained data were verified using qRT-PCR. Genes that were expressed differentially (DEGs) in two cell types were classified into functional groups and categories using different approaches. As a result, we portrayed physiological features that distinguish cross-protected phenotype from the growing cells.

Polyphenol oxidase from Pectobacterium atrosepticum : identification and cloning of gene and characteristics of the enzyme

In the present study, we attempted to elucidate if the harmful phytopathogenic bacteria of Pectobacterium genus (P. atrosepticum) possess the enzymes for oxidation of phenolic compounds. Polyphenol oxidase (laccase) activity was revealed in P. atrosepticum cell lysates. Using bioinformatic analysis, an ORF encoding a putative copper‐containing polyphenol oxidase of 241 amino acids with a predicted molecular mass of 25.9 kDa was found. This protein (named Pal1) shares significant level of identity with laccases of a new type described for several bacterial species. Cloning and expression of the pal1 gene and the analysis of corresponding recombinant protein confirmed that Pal1 possessed laccase activity. The recombinant Pal1 protein was characterized in terms of substrate specificity, kinetic parameters, pH and temperature optimum, sensitivity to inhibitors and metal content. Pal1 demonstrated alkali‐ and thermo‐tolerance. The kinetic parameters Km and kcat for 2,6‐dimethoxyphenol were 0.353 ± 0.062 mM and 98.79 ± 4.9 s−1, respectively. The protein displayed high tolerance to sodium azide, sodium fluoride, NaCl, SDS and cinnamic acid. The transcript level of the pal1 gene in P. atrosepticum was shown to be induced by plant‐derived phenolic compound (ferulic acid) and copper sulfate.

Recombinant maize 9-lipoxygenase: Expression, purification, and properties

Expression of maize 9-lipoxygenase was performed and optimized in Escherichia coli Rosetta(DE3)pLysS. The purity of recombinant protein obtained during Q-Sepharose and Octyl-Sepharose chromatographies in an LP system at 4°C was >95%. Maximum activity of the lipoxygenase reaction was observed at pH 7.5. Enzyme stability was studied at pH 4.5 to 9.5 and in the presence of different compounds: phenylmethanesulfonyl fluoride, β-mercaptoethanol, ammonium sulfate, and glycerol. HPLC and GC-MS analysis showed that enzyme produced 99% 9S-hydroperoxide from linoleic acid. 13-Hydroperoxide (less than 1%) consisted of S- and R-enantiomers in ratio 2 : 3.