Domain swapping localizes the structural determinants of regioselectivity in membrane-bound fatty acid desaturases of Caenorhabditis elegans.
Most fatty acid desaturases are members of a large superfamily of integral membrane, O(2)-dependent, iron-containing 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 omega-3 (FAT-1) and "Delta12" (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 nu+3, i.e. it "measures" three carbons from an existing double bond. The structural basis for nu+3 and omega-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 nu+3 to omega-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.
Sasata
Robert J
RJ
Plant Biotechnology Institute, Saskatoon, Saskatchewan S7N OW9, Canada.
Reed
Darwin W
DW
Loewen
Michèle C
MC
Covello
Patrick S
PS
D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans / Q000201:enzymology / Q000235:genetics*; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D044943:Fatty Acid Desaturases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D029742:Caenorhabditis elegans Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C466741:fat-1 protein, C elegans; C492490:fat-2 protein, C elegans; D044943:Fatty Acid Desaturases