Biochemical Characterization of the Oxygenation of Unsaturated Fatty Acids by the Dioxygenase and Hydroperoxide Isomerase of Pseudomonas aeruginosa 42A2

Ernst H. Oliw,Mats Hamberg,Angeles Manresa,Montse Busquets,Pilar Díaz,Eriel Martínez

doi:10.1074/jbc.m109.078147

Abstract

We have studied oxygenation of fatty acids by cell extract of Pseudomonas aeruginosa 42A2. Oleic acid ((9Z)-18:1) was transformed to (10S)-hydroperoxy-(8E)-octadecenoic acid ((10S)-HPOME) and to (7S,10S)-dihydroxy-(8E)-octadecenoic acid (7,10-DiHOME). Experiments under oxygen-18 showed that 7,10-DiHOME contained oxygen from air and was formed sequentially from (10S)-HPOME by isomerization. (10R)-HPOME was not isomerized. The (10S)-dioxygenase and hydroperoxide isomerase activities co-eluted on ion exchange chromatography and on gel filtration with an apparent molecular size of approximately 50 kDa. 16:1n-7, 18:2n-6, and 20:1n-11 were also oxygenated to 7,10-dihydroxy fatty acids, and (8Z)-18:1 was oxygenated to 6,9-dihydroxy-(7E)-octadecenoic acid. A series of fatty acids with the double bond positioned closer to ((6Z)-18:1, (5Z,9Z)-18:2) or more distant from the carboxyl group ((11Z)-, (13Z)-, and (15Z)-18:1) were poor substrates. The oxygenation mechanism was studied with [7S-(2)H]18:1n-9, [7R-(2)H]18:2n-6, and [8R-(2)H]18:2n-6 as substrates. The pro-R hydrogen at C-8 was lost in the biosynthesis of (10S)-HPODE, whereas the pro-S hydrogen was lost and the pro-R hydrogen was retained at C-7 during biosynthesis of the 7,10-dihydroxy metabolites. Analysis of the fatty acid composition of P. aeruginosa revealed relatively large amounts of (9E/Z)-16:1 and (11E/Z)-18:1 and only traces of 18:1n-9. We found that (11Z)-18:1 (vaccenic acid) was transformed to (11S,14S)-dihydroxy-(12E)-octadecenoic acid and to a mixture of 11- and 12-HPOME, possibly due to reverse orientation of (11Z)-18:1 at the active site compared with oleic acid. The reaction mechanism of the hydroperoxide isomerase suggests catalytic similarities to cytochrome P450.

Highlights

P. aeruginosa is an important opportunistic pathogen of immunocompromised patients, burn victims, and patients with cystic fibrosis [16, 17]
Preparation and purification of 1-mg amounts of the main enzymatic products in cultures of P. aeruginosa are described in the supplemental material. (10S)-HPOME was a poor substrate in comparison with oleic acid, whereas transformation of (10S)-HOME to the 7,10-diol could not be detected
We report that P. aeruginosa expresses a fatty acid diol synthase responsible for the sequential transformation of oleic acid to (10S)-HPOME and (7S,10-DiHOME is formed from (10S))-DiHOME

Summary

Introduction

P. aeruginosa is an important opportunistic pathogen of immunocompromised patients, burn victims, and patients with cystic fibrosis [16, 17]. In 1988, P. aeruginosa 42A2 was demonstrated to oxidize oleic acid to a new surfactant, a dihydroxy fatty acid metabolite [7]. This product was later identified as (7S,10S)-dihydroxy-(8E)-octadecenoic acid ((7S,10S)-DiHOME) by Hou and co-workers using the P. aeruginosa strain PR3 [8, 10, 15]. The genome of P. aeruginosa was sequenced in 2000, and this provides important information. Vance et al [21] found that P. aeruginosa secreted an arachidonate 15-lipoxygenase and expressed this protein by aid of the PA1169 sequence. As far as is known, lipoxygenases only oxidize oleic acid slowly [22]

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