A lipoxygenase with dual positional specificity is expressed in olives (Olea europaea L.) during ripening.

International audiencePlant lipoxygenases (LOXs) are a class of widespread dioxygenases catalysing the hydroperoxidation of polyunsaturated fatty acids. Although multiple isoforms of LOX have been detected in a wide range of plants, their physiological roles remain to be clarified. With the aim to clarify the occurrence of LOXs in olives and their contribution to the elaboration of the olive oil aroma, we cloned and characterized the first cDNA of the LOX isoform which is expressed during olive development. The open reading frame encodes a polypeptide of 864 amino acids. This olive LOX is a type-1 LOX which shows a high degree of identity at the peptide level towards hazelnut (77.3%), tobacco (76.3%) and almond (75.5%) LOXs. The recombinant enzyme shows a dual positional specificity, as it forms both 9- and 13-hydroperoxide of linoleic acid in a 2:1 ratio, and would be defined as 9/13-LOX. Although a LOX activity was detected throughout the olive development, the 9/13-LOX is mainly expressed at late developmental stages. Our data suggest that there are at least two Lox genes expressed in black olives, and that the 9/13-LOX is associated with the ripening and senescence processes. However, due to its dual positional specificity and its expression pattern, its contribution to the elaboration of the olive oil aroma might be considered

Expression et caractérisation de la lipoxygénase recombinante d'olive : une enzyme présentant une double spécificité d'hydroperoxydation et exprimée dans les derniers stades de développement des fruits.

Plant lipoxygenases (LOXs, EC 1.13.11.12) are a class of widespread dioxygenases catalyzing the hydroperoxidation of polyunsaturated fatty acids. Although multiple isoforms of LOX have been detected in a wide range of plants, their physiological roles remain to be clarified. With the aim to clarify the occurrence of LOXs in olives and their contribution to the elaboration of the olive oil aroma, we cloned and characterized the first cDNA of the LOX isoform which is expressed during olive development. The open reading frame encodes a polypeptide of 864 amino acids. This olive LOX is a type-1 LOX which shows a high degree of identity at the peptide level towards hazelnut (77.3%) and tobacco (76.3%). LOXs. The recombinant enzyme shows a dual positional specificity, as it releases both 9- and 13-hydroperoxide of linoleic acid in a 2:1 ratio. Although a LOX activity was detected throughout the olive development, the 9/13-LOX is mainly expressed at late developmental stages. Our data suggest that they are at least two Lox genes expressed in black olives, and that the 9/13-LOX is associated with the ripening and senescence processes. However, due to its dual positional specificity and its expression pattern, its contribution to the elaboration of the olive oil aroma might be considered. We modified substrate accessibility to olive LOX active site. Phe277 and Tyr280 were replaced by short chain residues (Ala and Ile) at the active site entrance. Those mutants (F277A, Y280I and F277A/Y280I) were expressed in E. coli and partially purified. A 4-fold conversion rate of linoleic acid was observed with F277A and F277A/Y280I mutants in comparison to recombinant olive LOX. Kinetic parameters of F277A and F277A/Y280I mutants were similar to soybean LOX1.Les lipoxygénases (LOXs, EC 1.13.11.12) sont des dioxygénases qui catalysent l'hydroperoxidation des acides gras polyinsaturés. Bien que de nombreuse isoformes de LOXs aient été caractérisé chez de nombreux végétaux, leur rôle physiologique reste inconnu. Dans le but d'éclaircir le rôle des LOXs dans l'olive et leur contribution dans l'élaboration de l'arôme, nous avons cloné et caractérisé un ADNc codant pour une isoforme de LOX d'olive exprimé dans les derniers stades de maturation du fruit. Cet ADNc présente un cadre de lecture ouvert qui code pour une protéine de 864 acides aminés. Cette LOX d'olive fait partie des LOXs de type-1 et présente un fort pourcentage d'identité avec la LOX de noisette (77,3%) et la LOX de tabac (76,3%). L'enzyme recombinante présente une double spécificité, elle produit des 9- et des 13-hydroperoxydes dans un rapport 2:1. Bien que l'activité LOX soit détectée à tous les stades de développement des olives, la 9/13-LOX est uniquement exprimé dans les stades noirs. Nos résultats suggèrent qu'il existe au moins deux gènes lox exprimés dans les olives noires, et que la 9/13-LOX est associé aux phénomènes de maturation et de sénescence. Cependant, en considérant sa double spécificité de substrat et son profil d'expression, nous ne pouvons pas exclure que cette enzyme puisse jouer un rôle dans l'élaboration de l'arôme. Nous avons modifié l'accessibilité au site actif de la LOX d'olive en mutant les résidus Phe277 et Tyr280 situés à l'entrée du site actif par des résidus à chaîne latérale courte (Ala et Ile). Nous avons exprimé les mutants F277A, Y280I et F277A/Y280I chez E. coli et nous les avons partiellement purifié. Les mutants F277A et F277A/Y280I présentent un taux de conversion de l'acide linoléique 4 fois supérieur à celui de la LOX d'olive non mutée et ont des paramètres cinétiques similaires à la LOX1 de soja

A novel lipoxygenase in olives (Olea europaea L.), with dual positional specificity, expressed late during fruit development

International audiencePlant lipoxygenases (LOXs) are a class of widespread dioxygenase catalyzing the hydroperoxidation of polyunsaturated fatty acids. Although multiple isoforms of LOX have been detected in a wide range of plants, their physiological role is still unclear. With the aim to clarify the occurrence of LOXs and their contribution to the elaboration of the olive oil aroma, we carried out the biochemical and molecular characterization of the LOX isoform expressed during olive development. A full-length cDNA was isolated by Reverse Transcription PCR and Rapid amplification of cDNA end carried out on total RNA from mature olives. The 2852 bp sequence displays an open reading frame of 2592 bp encoding a putative polypeptide of 864 amino acids with a calculated molecular mass of 98.4 kDa and a pI of 5.95. The olive LOX is a type-1 LOX and shows a high degree of identity towards hazelnut (77.3%), tobacco (76.3%) and almond (75.5%) LOXs. The recombinant enzyme produced in E. coli shows a dual positional specificity, as it releases both 9- and 13-hydroperoxide of linoleic acid in a 2:1 ratio. Although LOX activity was detected throughout the olive development, the 9/13-LOX is mainly expressed at late developmental stages. Our data suggest the presence of at least two LOX isoforms in black olives, and that the 9/13-LOX is associated with the ripening and senescence processes. However, due to its dual positional specificity ant its expression pattern, its contribution to the elaboration of the olive oil aroma might be considered

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