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.

Most fatty acid desaturases are members of a large superfamily of integral membrane, O 2 -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 ؉3 to -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.Fatty acid desaturases are part of multicomponent systems that catalyze the oxygen-and nicotinamide adenine dinucleotidedependent syn-dehydrogenation of unactivated aliphatic regions of their fatty ester (acyl-lipid) or thioester (acyl-acyl carrier protein or acyl-CoA) substrates (1-6). In addition to the family of soluble fatty acid desaturases, of which the plant stearoyl-acyl carrier protein desaturase is well characterized, there is a large group of structurally distinct integral membrane desaturases. Membrane desaturases of widely varying substrate specificity and regioselectivity are scattered among a range of taxa. The yeast Saccharomyces cerevisiae has a single stearoyl-CoA ⌬9 desaturase, whereas many bacteria lack such desaturases entirely. At the other extreme, the biosynthesis of (4Z,7Z,10Z,13Z,16Z,19Z)-docosahexaenoic acid in some marine fungi likely requires the successive action of six structurally similar membrane desaturases, each varying in substrate specificity and regioselectivity (7,8).Based on structural and catalytic similarities, membrane desaturases are included in a superfamily of oxidative enzymes along with alkane hydroxylase, xylene monooxygenase, carotene ketolase, and sterol methyloxidase (9, 10). These are thought to contain a histidine-coordinated diiron center at the active site. While essentially no three-dimensional structural information is available for these difficult to purify enzymes, primary structure similarity, especially the conservation of three histidine-rich motifs, and similarity o...

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