It is estimated that plants contain thousands of fatty acid structures, many of which arise by the action of membrane-bound desaturases and desaturase-like enzymes. The details of ''unusual'' e.g., hydroxyl or conjugated, fatty acid formation remain elusive, because these enzymes await structural characterization. However, soluble plant acyl-ACP (acyl carrier protein) desaturases have been studied in far greater detail but typically only catalyze desaturation (dehydrogenation) reactions. We describe a mutant of the castor acyl-ACP desaturase (T117R/G188L/D280K) that converts stearoyl-ACP into the allylic alcohol trans-isomer (E)-10-18:1-9-OH via a cis isomer (Z)-9-18:1 intermediate. The use of regiospecifically deuterated substrates shows that the conversion of (Z)-9-18:1 substrate to (E)-10-18:1-9-OH product proceeds via hydrogen abstraction at C-11 and highly regioselective hydroxylation (>97%) at C-9. 18 O-labeling studies show that the hydroxyl oxygen in the reaction product is exclusively derived from molecular oxygen. The mutant enzyme converts (E)-9-18:1-ACP into two major products, (Z)-10-18:1-9-OH and the conjugated linolenic acid isomer, (E)-9-(Z)-11-18:2. The observed product profiles can be rationalized by differences in substrate binding as dictated by the curvature of substrate channel at the active site. That three amino acid substitutions, remote from the diiron active site, expand the range of reaction outcomes to mimic some of those associated with the membrane-bound desaturase family underscores the latent potential of O2-dependent nonheme diiron enzymes to mediate a diversity of functionalization chemistry. In summary, this study contributes detailed mechanistic insights into factors that govern the highly selective production of unusual fatty acids.binuclear iron ͉ diiron ͉ hydroxylation ͉ nonheme iron ͉ catalysis

The stereochemistry of palmitoyl-ACP Delta(4) desaturase-mediated dehydrogenation has been examined by tracking the fate of deuterium atoms located on stereospecifically monodeuterated substrates-(4S)- and (4R)-[4-(2)H(1)]-palmitoyl-ACP and (5S)- and (5R)-[5-(2)H(1)]-palmitoyl-ACP. It was found that the introduction of the (Z)-double bond between C-4 and C-5 of a palmitoyl substrate occurs with pro-R enantioselectivity-a result which matches that obtained for a closely related homolog-castor stearoyl-ACP Delta(9) desaturase. These data show that despite the difference in regioselectivity between the two enzymes, the stereochemistry of hydrogen removal is conserved.

The stereochemistry of castor stearoyl-ACP Delta(9) desaturase-mediated 10-sulfoxidation has been determined. This was accomplished by (19)F NMR analysis of a fluorine-tagged product, 18-fluoro-10-thiastearoyl ACP S-oxide, in combination with a chiral solvating agent, (R)-AMA. Sulfoxidation proceeds with the same stereoselectivity as hydrogen removal from the parent stearoyl substrate. These data validate the use of thia probes to determine the stereochemistry and cryptoregiochemistry of desaturase-mediated oxidations.

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1H-decoupled 19F NMR has been used to monitor the highly regioselective oxidation of a fluorine-tagged thia-fatty acid derivative by castor stearoyl-ACP delta9 desaturase. The major enzymatic product, after reductive work-up, was identified as 9-fluoro-1-nonanol. This compound could be easily distinguished from substrate and a 9-sulfoxy by-product on the basis of its 19F NMR chemical shift and spiking experiments using authentic standards. Structural assignment of the cleavage product was confirmed by GC-MS analysis of the enzymatic products.