Unveiling the genes responsible for the unique Pseudomonas aeruginosa oleate-diol synthase activity

• Published in: Biochimica et biophysica acta 2014-10, Vol.1841 (10), p.1360-1371
• Main Authors: Estupiñán, Mónica, Diaz, Pilar, Manresa, Angels
• Format: Article
• Language: English
• Subjects: Oleate-diol synthase activity; Oleic acid metabolism; Oxylipin; P. aeruginosa
• ISSN: 1388-1981; 0006-3002; 1879-2618
• DOI: 10.1016/j.bbalip.2014.06.010

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 ((7S,10S)-dihydroxy-(8E)-octadecenoic acid), both of which accumulate in the culture supernatant. Several mutants of P. aeruginosa PAO1 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 acids, 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 a new fine regulated oleate-diol synthase operon, originated by a gene duplication event followed by neofunctionalization for environmental adaptation, being unprecedented in prokaryotes. •Rational approach used to identify P. aeruginosa oleate diol synthase-coding genes•OA is converted into 7,10-DiHOME by dioxygenase PA2077 and diol synthase PA2078.•Genes PA2077 and PA2078, tandemly located, constitute a bicistronic operon.•No heterocomplex formation is required for the activity of the individual enzymes.•Probably, a duplication event followed by neofunctionalization generated the operon.