期刊
BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR AND CELL BIOLOGY OF LIPIDS
卷 1841, 期 10, 页码 1360-1371出版社
ELSEVIER
DOI: 10.1016/j.bbalip.2014.06.010
关键词
P. aeruginosa; Oleate-diol synthase activity; Oxylipin; Oleic acid metabolism
资金
- Ministerio de Economia y Competitividad (CICYT) [CTQ2010-21183-CO2-01/02]
- IV Pla de Recerca de Catalunya (Generalitat de Catalunya) [2009SGR-819]
- Xarxa de Referencia en Biotecnologia [Biotecnologia XRB-2010]
- Comissionat per a Universitats i Recerca (CUR) of Departament d'Innovacio, Universitats i Empresa (DIUE) from Generalitat de Catalunya and European Social Source [FI-DGR2011]
- NIH for P. aeruginosa PAO1 Transposon Mutant collection (UWGC) [P30 DK089507]
Pseudomonas aeruginosa displays the ability to perform bioconversion of oleic acid into a class of hydroxylated fatty acids known as oxylipins. A diol synthase activity is responsible for such a conversion, which proceeds through the dioxygenation of oleic acid to release hydroperoxide 10-H(P)OME ((10S)-hydroxy-(8E)-octadecenoic acid), followed by conversion of the hydroperoxide intermediate into 7,10-DiHOME ((75,10S)-dihydroxy-(8E)-octadecenoic acid), both of which accumulate in the culture supernatant. Several mutants of P. aeruginosa PAW were analyzed for the production of 10-H(P)OME and 7,10-DiHOME and two of them (ORFs PA2077 and PA2078), unable to release hydroxylated fatty adds, were detected and selected for further analysis. Involvement of ORFs PA2077 and PA2078 in oleate-diol synthase activity was confirmed, and their respective role in the conversion of oleic acid was analyzed by mutation complementation. Activity restoration revealed that gene PA2077 codes for the 10S-dioxygenase activity (10S-DOX) responsible for the first step of the reaction, whereas PA2078 encodes for the (7S,10S)-hydroperoxide diol synthase enzyme (7,10-DS) which allows the conversion of 10-H(P)OME into 7,10-DiHOME. Heterologous expression of both enzymes separately showed that no hetero-complex formation is required for enzymatic activity. Bioinformatics and RT-PCR analysis revealed that both genes constitute anew fine regulated oleate-diol synthase operon, originated by a gene duplication event followed by neofunctionalization for environmental adaptation, being unprecedented in prokaryotes. (C) 2014 Elsevier B.V. All rights reserved.
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