RESUMO
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.
Assuntos
Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Ácidos Graxos Dessaturases/química , Ácidos Graxos Dessaturases/genética , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/enzimologia , Proteínas de Caenorhabditis elegans/metabolismo , Ácidos Graxos Dessaturases/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The mechanism by which the fatty acid acetylenase of Crepis alpina catalyzes crepenynic acid ((9Z)-octadeca-9-en-12-ynoic acid) production from linoleic acid has been probed through the use of kinetic isotope effect (KIE) measurements. This was accomplished by incubating appropriate mixtures of regiospecifically deuterated isotopomers with a strain of Saccharomyces cerevisiae expressing a functional acetylenase. LC/MS analysis of crepenynic acid obtained in these experiments showed that the oxidation of linoleate occurs in two discrete steps, since the cleavage of the C12-H bond is very sensitive to isotopic substitution (k(H)/k(D) = 14.6 +/- 3.0) while a minimal isotope effect (k(H)/k(D) = 1.25 +/- 0.08) was observed for the C13-H bond breaking step. These data suggest that crepenynic acid is produced via initial H-atom abstraction at C12 of a linoleoyl substrate. The relationship between the mechanism of enzymatic acetylenation and epoxidation is discussed.
