A 49-kDa mini-lipoxygenase from Anabaena sp. PCC 7120 retains catalytically complete functionality

Abstract

Anabaena sp. PCC 7120 is one of the few prokaryotes harboring a lipoxygenase (LOX) gene. The sequence resides in an open reading frame encoding a fusion protein of a catalase-like hemoprotein with an unusually short LOX (approximately 49 kDa) at the C terminus. The recombinant mini-LOX contains a non-heme iron in the active site and is highly active with linoleic and alpha-linolenic acids (which occur naturally in Anabaena) giving the respective 9R-hydroperoxides, the mirror image of the 9S-LOX products of plants. Using stereospecifically labeled [11-(3)H]linoleic acids we show that reaction is catalyzed via a typical antarafacial relationship of initial hydrogen abstraction and oxygenation. The mini-LOX oxygenated C16/C18:2-phosphatidylcholine with 9R specificity, suggesting a "tail first" mode of fatty acid binding. Site-directed mutagenesis of an active site Ala (Ala215), typically conserved as Gly in R-LOX, revealed that substitution with Gly retained 9R specificity, whereas the larger Val substitution switched oxygenation to 13S, implying that Ala215 represents the functional equivalent of the Gly in other R-LOX. Metabolism studies using a synthetic fatty acid with extended double bond conjugation, 9E,11Z,14Z-20:3omega6, showed that the mini-LOX can control oxygenation two positions further along the fatty acid carbon chain. We conclude that the mini-LOX, despite lacking the beta-barrel domain and much additional sequence, is catalytically complete. Interestingly, animal and plant LOX, which undoubtedly share a common ancestor, are related in sequence only in the catalytic domain; it is possible that the prokaryotic LOX represents a common link and that the beta-barrel domain was then acquired independently in the animal and plant kingdoms.