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J. Biol. Chem., Vol. 279, Issue 38, 39296-39302, September 17, 2004

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Domain Swapping Localizes the Structural Determinants of Regioselectivity in Membrane-bound Fatty Acid Desaturases of Caenorhabditis elegans^*

Robert J. Sasata, Darwin W. Reed, Michèle C. Loewen, and Patrick S. Covello

From the Plant Biotechnology Institute, Saskatoon, Saskatchewan S7N OW9, Canada

Most fatty acid desaturases are members of a large superfamily of integral membrane, O₂-dependent, ironcontaining enzymes that catalyze a variety of oxidative modifications to lipids. Sharing a similar primary structure and membrane topology, these enzymes are broadly categorized according to their positional specificity or regioselectivity, which designates the preferred position for substrate modification. To investigate the structural basis of regioselectivity in membrane-bound desaturases, the Caenorhabditis elegans -3 (FAT-1) and "12" (FAT-2) desaturases were used as a model system. With the use of unnatural substrates, the regioselectivity of C. elegans FAT-2 was clearly defined as +3, i.e. it "measures" three carbons from an existing double bond. The structural basis for +3 and -3 regioselectivities was examined through construction and expression of chimeric DNA sequences based on FAT-1 and FAT-2. Each sequence was divided into seven domains, and chimeras were constructed in which specific domains were replaced with sequence from the other desaturase. When tested by expression in yeast using exogenously supplied substrates, chimeric sequences were found in which domain swapping resulted in a change of regioselectivity from +3to -3 and vice versa. In this way, the structural determinants of regioselectivity in FAT-1 and FAT-2 have been localized to two interdependent regions: a relatively hydrophobic region between the first two histidine boxes and the carboxyl-terminal region.

Received for publication, May 24, 2004 , and in revised form, June 28, 2004.

^* This work was supported by the Natural Sciences and Engineering Research Council of Canada (to P. S. C.), the National Research Council of Canada (to P. S. C., M. C. L., and R. J. S.), and the University of Saskatchewan (to R. J. S.). 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.

To whom correspondence should be addressed: National Research Council, Plant Biotechnology Inst., 110 Gymnasium Place, Saskatoon, Saskatchewan S7N 0W9, Canada. Tel.: 306-975-5269; Fax: 306-975-4839; E-mail: Patrick.Covello{at}nrc-cnrc.gc.ca.

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