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J. Biol. Chem., Vol. 280, Issue 47, 39545-39552, November 25, 2005

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Insights from the X-ray Crystal Structure of Coral 8R-Lipoxygenase

CALCIUM ACTIVATION VIA A C2-LIKE DOMAIN AND A STRUCTURAL BASIS OF PRODUCT CHIRALITY^*

Michael L. Oldham, Alan R. Brash¶, and Marcia E. Newcomer1

From the Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803 and the Departments of Biochemistry and ^¶Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

Lipoxygenases (LOXs) catalyze the regio- and stereospecific dioxygenation of polyunsaturated membrane-embedded fatty acids. We report here the 3.2 Å resolution structure of 8R-LOX from the Caribbean sea whip coral Plexaura homomalla, a LOX isozyme with calcium dependence and the uncommon R chiral stereospecificity. Structural and spectroscopic analyses demonstrated calcium binding in a C2-like membrane-binding domain, illuminating the function of similar amino acids in calcium-activated mammalian 5-LOX, the key enzyme in the pathway to the pro-inflammatory leukotrienes. Mutation of Ca²⁺-ligating amino acids in 8R-LOX resulted not only in a diminished capacity to bind membranes, as monitored by fluorescence resonance energy transfer, but also in an associated loss of Ca²⁺-regulated enzyme activity. Moreover, a structural basis for R chiral specificity is also revealed; creation of a small oxygen pocket next to Gly⁴²⁸ (Ala in all S-LOX isozymes) promoted C-8 oxygenation with R chirality on the activated fatty acid substrate.

Received for publication, June 20, 2005 , and in revised form, September 7, 2005.

The atomic coordinates and structure factors (code 1zq4) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported in part by National Institutes of Health Grants GM 55420 (to M. E. N.) and GM 53638 (to A. R. B.) and by the Louisiana Governor's Biotechnology Initiative. 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.

¹ To whom correspondence should be addressed: Dept. of Biological Sciences, 202 Life Sciences Bldg., Louisiana State University, Baton Rouge, LA 70803. Tel.: 225-578-7383; Fax: 225-578-7258; E-mail: newcomer{at}lsu.edu.

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