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

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

Activation and Stabilization of Olive Recombinant 13-Hydroperoxide Lyase Using Selected Additives

International audienc

L'olivier : les ressources génétiques

Correspondance: [email protected] audienceLa génétique nous éclaire sur le passé mais prépare aussi l'avenir. Il existe dans le monde 2000 variétés d'olivier, 150 en France. Elles peuvent être identifiées selon leurs caractères morphologiques, la description portant sur l'arbre, ses feuilles, ses fleurs, ses olives et leurs noyaux. Mais cette méthode n'est pas suffisante pour caractériser chacune des variétés d'où une nouvelle approche complémentaire à l'aide de marqueurs basés sur le polymorphisme de l'ADN. Ces marqueurs génétiques ont également permis de reconstituer l'histoire des origines de l'olivier, de l'olivier sauvage, l'oléastre, à l'olivier cultivé. Des travaux de sélection ont été entrepris à l'INRA depuis de longues années et se poursuivent aujourd'hui avec des collaborations internationales

The European “Clinical Trial” Regulation; Relationship with the Jardé Act: a Giens Workshop

In May 2014, the European Union Parliament and Council published a new regulation on clinical trials on medicinal products for human use, which is designed to replace Directive 2001/20/EC. It will not come into effect until 2016. Nevertheless, it is essential to examine its relationship with national legislation, i.e. the Jardé Act, whose implementation has been delayed pending publication of the European regulation. The Giens workshop identified and examined the various issues that this relationship is bound to raise. In particular, it looked at trial methodology assessment procedures, the working relationship between the French National Agency of Drug Safety and Health Products (Agence Nationale de Sécurité du Médicament et des Produits de Santé, ANSM) and ethics committees during the authorization application evaluation phase, review of post-authorization/registration studies on medicinal products and medical devices, and data transparency. Abbreviations: see end of article

Les produits et les usages de l’olivier

Correspondance : [email protected] audienceLes olives et l'huile d'olive sont des produits bien connus de l'olivier. L'huile d'olive est un aliment emblématiqe dans toute la Méditerranée. Des recherches génétiques concernent sa traçabilité. Les signes de qualité se développent, notamment les AOC. Dans les temps anciens, elle est source d'éclairage. De tous temps, elle entre dans la fabrication de parfums, de savons renommés de Marseille, d'Alep... Elle est aussi riche en composants: acide oléique et polyphénols, qui diminuent les risques cardio-vasculaires ou l'ostéoporose. Des substances provenant de l'olivier et de l'huile d'olive entrent dans la composition de produits pharmaceutiques. La valorisation d'autres sous-produits est plus inattendue: dans les pays du sud de la Méditerranée, les grignons dont on a retiré les coques ainsi que des feuilles et des brindilles de l'arbre sont donnés comme aliments au bétail; les coques et les coupes de bois sont utilisées comme sources d'énergie; les margines peuvent devenir des fertilisant