using lipid profiles and genotypes for the characterization of corsican olive oils

International audiencein 2004, ...

Changes in organic acids and sugars during early stages of development of acidic and acidless citrus fruit

International audienceMost of the studies on organic acids and sugars in citrus were performed during fruit maturation, and less is known before this stage of development. The aim of our study was to investigate acids and sugars in lemon, lime, and orange from fruit-set toward development. We chose to compare organic acid and sugar accumulation among acidic and acidless varieties within three species. We estimated the acidity by titrimetry and quantified the concentrations of seven organic acids and three sugars by reverse HPLC. During the first 50 days of development, quinic acid was the major organic acid whatever the variety. Afterward, citric acid predominated in acidic varieties, while in acidless, malic acid exceeded it. Fructose substituted citric acid in acidless and could be synthesized either from citric acid or directly from glucose. Our results provided the first complete report on sugar and organic acid accumulation during the early stages of fruit development in several citrus varieties

A functional role identified for conserved charged residues at theactive site entrance of lipoxygenase with double specificity

International audiencePlant lipoxygenases (LOXs) are a class of widespread dioxygenases catalyzing the hydroperoxidation offree polyunsaturated fatty acids, producing 9-hydroperoxides or 13-hydroperoxides from linoleic and-linolenic acids, and are called 9-LOX or 13-LOX, respectively. Some LOXs produce both 9- and 13-hydroperoxides. The models proposed to explain the reaction mechanism specificity fail to explain the“double specificity” character of these LOXs. In this study, we used the olive LOX1 with double specificityto investigate the implication of the charged residues R265, R268, and K283 in the orientation of thesubstrate into the active site. These residues are present in a conserved pattern around the entrance ofthe active site. Our results show that these residues are involved in the penetration of the substrate intothe active site: this positive patch could capture the carboxylate end of the substrate, and then guide itinto the active site. Due to its position on 2 helix, the residue K283 could have a more important role, itsinteraction with the substrate facilitating the motions of residues constituting the “cork of lipoxygenases”or the 2 helix, by disrupting putative hydrogen and ionic bonds

A functional role identified for conserved charged residues at theactive site entrance of lipoxygenase with double specificity

International audiencePlant lipoxygenases (LOXs) are a class of widespread dioxygenases catalyzing the hydroperoxidation offree polyunsaturated fatty acids, producing 9-hydroperoxides or 13-hydroperoxides from linoleic and-linolenic acids, and are called 9-LOX or 13-LOX, respectively. Some LOXs produce both 9- and 13-hydroperoxides. The models proposed to explain the reaction mechanism specificity fail to explain the“double specificity” character of these LOXs. In this study, we used the olive LOX1 with double specificityto investigate the implication of the charged residues R265, R268, and K283 in the orientation of thesubstrate into the active site. These residues are present in a conserved pattern around the entrance ofthe active site. Our results show that these residues are involved in the penetration of the substrate intothe active site: this positive patch could capture the carboxylate end of the substrate, and then guide itinto the active site. Due to its position on 2 helix, the residue K283 could have a more important role, itsinteraction with the substrate facilitating the motions of residues constituting the “cork of lipoxygenases”or the 2 helix, by disrupting putative hydrogen and ionic bonds

genetic diversity in tunisian olive accessions and their relatedness with other mediterranean olive genotypes

International audienceeighty-four ....

Identification of putative residues involved in the accessibility of the substrate-binding site of lipoxygenase by site-directed mutagenesis studies.

International audienceLipoxygenases (LOXs) are a class of widespread dioxygenases catalyzing the hydroperoxidation of polyunsaturated fatty acids (PUFA). Recently, we isolated a cDNA encoding a LOX, named olive LOX1, from olive fruit of which the deduced amino acid sequence shows more than 50% identity with plant LOXs. In the present study, a model of olive LOX1 based on the crystal structure of soybean LOX-1 as template has been generated and two bulky amino acid residues highly conserved in LOXs (Phe277) and in plant LOXs (Tyr280), located at the putative entrance of catalytic site were identified. These residues may perturb accessibility of the substrate-binding site and therefore were substituted by less space-filling residues. Kinetic studies using linoleic and linolenic acids as substrates were carried out on wild type and mutants. The results show that the removal of steric bulk at the entrance of the catalytic site induces an increase of substrate affinity and of catalytic efficiency, and demonstrate that penetration of substrates into active site of olive LOX1 requires the movement of the side chains of the Phe277 and Tyr280 residues. This study suggests the involvement of these residues in the accessibility of the substrate-binding site in the lipoxygenase family

Olive Recombinant Hydroperoxide Lyase, an Efficient Biocatalyst for Synthesis of Green Leaf Volatiles

International audienceVolatile C6-aldehydes are the main contributors to the characteristic odor of plantsknown as Bgreen note^ and are widely used by the flavor industry. Biotechnological processeswere developed to fulfill the high demand in C6-aldehydes in natural flavorants and odorants.Recombinant hydroperoxide lyases (HPLs) constitute an interesting alternative to overcomedrawbacks arising from the use of HPL from plant extracts. Thus, olive recombinant 13-HPLwas assayed as biocatalysts to produce C6-aldehydes. Firstly, a cDNA encoding for olive HPLof Leccino variety was isolated and cloned in pQE-30 expression vector. In order to improvethe enzyme solubility, its chloroplast transit peptide was deleted. Both enzymes (HPL wild typeand HPL deleted) were expressed into Escherichia coli strain M15, purified, characterized, andthen used for bioconversion of 13-hydroperoxides of linoleic and linolenic acids. Aldehydesproduced were extracted, then identified and quantified using gas chromatography and massspectrometry. Recombinant HPL wild type (HPLwt) allowed producing 5.61 mM of hexanaland 4.39 mM of 3Z-hexenal, corresponding to high conversion yields of 93.5 and 73 %,respectively. Using HPL deleted (HPLdel) instead of HPLwt failed to obtain greater quantitiesof hexanal or 3Z-hexenal. No undesirable products were formed, and no isomerization of 3Zhexenalin 2E-hexenal occurred. The olive recombinant HPLwt appears to be a promisingefficient biocatalyst for the production of C6-aldehyde