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J. Biol. Chem., Vol. 283, Issue 8, 5138-5147, February 22, 2008

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A 49-kDa Mini-lipoxygenase from Anabaena sp. PCC 7120 Retains Catalytically Complete Functionality^*

From the Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

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 (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 -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 [11-³H]linoleic acids we show that reaction is catalyzed via a typical antarafacial relationship of initial hydrogen abstraction and oxygenation. The mini-LOX oxygenated C₁₆/C_18:2-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-20:36, 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 β-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 β-barrel domain was then acquired independently in the animal and plant kingdoms.

Received for publication, July 13, 2007 , and in revised form, October 24, 2007.

^* This work was supported by National Institutes of Health Grant GM-074888. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S5.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Pharmacology, Vanderbilt University School of Medicine, 23rd Ave. S. at Pierce, Nashville, TN 37232-6602. Tel.: 615-343-4495; Fax: 615-322-4707; E-mail: alan.brash{at}vanderbilt.edu.

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