Lipoxygenases are non-heme iron enzymes essential in eukaryotes, where they catalyze formation of the fatty acid hydroperoxides that are required by diverse biological (and also pathological) processes. In prokaryotes, most of them totally lacking in polyunsaturated fatty acids, the possible biological roles of lipoxygenases have remained obscure.To date, the available structures of lipoxygenases were only of eukaryotic origin. Here we report the first crystal structure of a prokaryotic lipoxygenase, from Pseudomonas aeruginosa (Pa_LOX). The structure shows major differences with respect to the eukaryotic enzymes, while retaining much of the overall lipoxygenase fold. The most striking difference is an insertion in the catalytic domain of a pair of long antiparallel alpha-helices, contributing to an enlarged binding pocket in Pa_LOX containing a bound phospholipid: a phosphatidylethanolamine with well defined chains of 18 and 14 carbons in length. The insertion acts as a lid that apparently needs to be opened to allow entrance of the phospholipid into the substrate-binding pocket. This idea is supported by the variability observed for this two-helix motif in a second crystal form and by the flexibility of the residues linking the motif to the protein. The mobility of the two-helix motif, together with the specific interactions observed between the phospholipid polar head and the protein residues, suggest a mechanism by which the enzymatic activity of secreted Pa_LOX can be exerted directly on target cell membranes.