RESUMEN
A novel CYP74 clan gene CYP443С1 of the starlet sea anemone (Nematostella vectensis, Cnidaria) has been cloned, and the properties of the corresponding recombinant protein have been studied. Depending on the substrate, CYP443С1 exhibited double function hydroperoxide lyase/epoxyalcohol synthase activity.
Asunto(s)
Aldehído-Liasas/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Anémonas de Mar/enzimología , Aldehído-Liasas/química , Aldehído-Liasas/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/genética , Anémonas de Mar/genética , Alineación de SecuenciaRESUMEN
Cytochromes P450 of the CYP74 family play a key role in the lipoxygenase cascade generating oxylipins (products of polyunsaturated fatty acid oxidation). The CYP74 family includes allene oxide synthases, hydroperoxide lyases, divinyl ether synthases, and epoxyalcohol synthases. In this work, we cloned the CYP74A88 gene from the Japanese buttercup (Ranunculus japonicus) and studied the properties of the encoded recombinant protein. The CYP74A88 enzyme specifically converts linoleic acid 9- and 13-hydroperoxides to oxiranyl carbinols 9,10-epoxy-11-hydroxy-12-octadecenoic acid and 11-hydroxy-12,13-epoxy-9-octadecenoic acid, respectively, which was confirmed by GC-MS analysis and kinetic studies. Therefore, the CYP74A88 enzyme is a specific epoxyalcohol synthase.
Asunto(s)
Biocatálisis , Clonación Molecular , Sistema Enzimático del Citocromo P-450/metabolismo , Ranunculus/enzimología , Sistema Enzimático del Citocromo P-450/genética , Cromatografía de Gases y Espectrometría de Masas , JapónRESUMEN
Data on the influence of the double bond geometry on the antimicrobial properties of different isomers of etherolenic acid against phytopathogenic bacteria are presented. (ω5Z)-Etherolenic acid possesses bactericidal properties against Xanthomonas campestris ssp. vesicatoria, Pseudomonas syringae ssp. tomato, Pectobacterium atrosepticum SCRI1043; the etherolenic and (11Z)-etherolenic acids possess only bacteriostatic properties.
Asunto(s)
Antibacterianos , Bacterias/crecimiento & desarrollo , Ácidos Grasos Insaturados , Lipooxigenasa , Proteínas de Plantas , Antibacterianos/química , Antibacterianos/farmacología , Ácidos Grasos Insaturados/química , Ácidos Grasos Insaturados/farmacología , EstereoisomerismoRESUMEN
The site-directed mutagenesis of tomato allene oxide synthase LeAOS3 (CYP74C3) on the "F/L toggle" site resulted in a partial conversion of LeAOS3 into epoxyalcohol synthase.
Asunto(s)
Sistema Enzimático del Citocromo P-450 , Oxidorreductasas Intramoleculares , Proteínas de Plantas , Solanum lycopersicum , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/genética , Oxidorreductasas Intramoleculares/química , Oxidorreductasas Intramoleculares/genética , Solanum lycopersicum/enzimología , Solanum lycopersicum/genética , Proteínas de Plantas/química , Proteínas de Plantas/genéticaAsunto(s)
Sustitución de Aminoácidos , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Sistema Enzimático del Citocromo P-450/metabolismo , Proteínas de Plantas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Dominio Catalítico , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/genética , Proteínas de Plantas/química , Proteínas de Plantas/genéticaRESUMEN
The in vitro metabolism of [1-(14)C]linoleate, [1-(14)C]linolenate and their 9(S)-hydroperoxides was studied in cell-free preparations from tulip (Tulipa gesneriana) bulbs, leaves and flowers. Linoleate and its 9-hydroperoxide were converted by bulb and leaf preparations into three ketols: (12Z)-9-hydroxy-10-oxo-12-octadecadienoic acid (alpha-ketol), (11E)-10-oxo-13-hydroxy-11-octadecadienoic acid (gamma-ketol) and a novel compound, (12Z)-10-oxo-11-hydroxy-12-octadecadienoic acid (10,11-ketol), in the approximate molar proportions of 10:3:1. The corresponding 15, 16-dehydro alpha- and gamma-ketols were the main metabolites of [1-(14)C]linolenate and its 9-hydroperoxide. Thus bulbs and leaves possessed 9-lipoxygenase and allene oxide synthase activities. Incubations with flower preparations gave alpha-ketol hydro(pero)xides as predominant metabolites. Bulb and leaf preparations possessed a novel enzyme activity, gamma-ketol reductase, which reduces gamma-ketol to 10-oxo-13-hydroxyoctadecanoic acid (dihydro-gamma-ketol) in the presence of NADH. Exogenous linolenate 13(S)-hydroperoxide was converted mostly into chiral (9S,13S)-12-oxo-10-phytodienoate (99.5% optical purity) by bulb preparations, while [1-(14)C]linolenate was a precursor for ketols only. Thus tulip bulbs possess abundant allene oxide cyclase activity, the substrate for which is linolenate 13(S)-hydroperoxide, even though 13(S)-lipoxygenase products were not detectable in the bulbs. The majority of the cyclase activity was found in the microsomes (10(5) g pellet). Cyclase activity was not found in the other tissues examined, but only in the bulbs. The ketol route of the lipoxygenase pathway, mediated by 9-lipoxygenase and allene oxide synthase activities, has not been detected previously in the vegetative organs of any plant species.
Asunto(s)
Lipooxigenasa/metabolismo , Magnoliopsida/enzimología , Radioisótopos de Carbono , Cromatografía Líquida de Alta Presión , Ácidos Grasos/metabolismo , Ácido Linoleico/metabolismo , Ácidos Linolénicos/metabolismo , Peróxidos Lipídicos/metabolismo , Espectrometría de Masas , Espectrofotometría UltravioletaRESUMEN
The metabolism in vitro of [1-(14)C]linoleate, [1-(14)C]linolenate and their 9(S)-hydroperoxides in tulip (Tulipa gesneriana) was found to be under the control of 9-lipoxygenase and allene oxide synthase, and directed towards alpha-ketol, gamma-ketol and the novel compound (12Z)-10-oxo-11-hydroxy-12-octadecadienoic acid (10,11-ketol). Potent activity of allene oxide cyclase (in bulbs) and a new enzyme, gamma-ketol reductase (in bulbs and leaves), was detected. Metabolism in flowers is directed predominantly towards alpha-ketol hydroperoxide.
Asunto(s)
Liliaceae/enzimología , Ácido Linoleico/metabolismo , Peróxidos Lipídicos/metabolismo , Lipooxigenasa/metabolismo , Fitosteroles/biosíntesis , Ácido alfa-Linolénico/metabolismo , Radioisótopos de Carbono , Oxidorreductasas Intramoleculares/metabolismo , Hojas de la Planta/enzimología , Raíces de Plantas/enzimología , Tallos de la Planta/enzimologíaRESUMEN
Incubations of [1-14C]linoleic acid or [1-14C]-(9Z,11E, 13S)-13-hydropero xy-9,11-octadecadienoic acid (13-HPOD) with juice of garlic bulbs lead to the formation of one predominant labelled product, viz., the novel divinyl ether (9Z,11E, 1'E)-12-(1'-hexenyloxy)-9,11-dodecadienoic acid ('etheroleic acid'). With lesser efficiency [1-14C]alpha-linolenic acid or [1-14C](9Z,11E, 13S,15Z)-13-hydroperoxy-9,11,15-octadecatrienoic acid (13-HPOT) are converted in this way into (9Z,11E,1'E,1'E,3'Z)-12-(1',3'-hexadienyloxy)-9,11- dodecadienoic acid ('etherolenic acid'). Thus, garlic bulbs possess the activity of a new 13-hydroperoxide-specific divinyl ether synthase.