Revealing the catalytic potential of an acyl-ACP desaturase: Tandem selective oxidation of saturated fatty acids

Abstract

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

Remote control of regioselectivity in acyl-acyl carrier protein-desaturases

“…The construction of mutant Asp280Lys was previously described (31) and the other Asp280 mutants were made using the same approach. Mutant Asp280Ala was further mutagenized to convert Ala284 to either an Asp or Lys.…”

“…The hexane was evaporated by incubation at room temperature overnight to a final volume of approximately 100 μL in preparation for GC analysis. Analysis was performed with the use of an HP5973 gas chromatograph-mass spectrometer (Hewlett-Packard) fitted with 60-m × 250-μm SP-2340 capillary columns (Supelco) as previously described (31). Assignments of double-bond position were based on elution times of methyl esters and identities confirmed by MS analysis of dimethyl disulfide derivatives, performed as previously described (34).…”

“…Mutant Asp280Ala was further mutagenized to convert Ala284 to either an Asp or Lys. Fatty acid desaturase reactions were performed by incubation of the desaturase with 14∶0-ACP substrate in the presence of recombinant spinach ACP-I (32) as previously described (31,33). Fatty acid methyl esters (FAMEs) were prepared by addition of 2 mL of 1% NaOCH 3 in methanol and incubation for 60 min at 50°C, after which the mixture was acidified with glacial acetic acid and the FAMEs extracted into hexane.…”

Desaturases: Emerging Models for Understanding Functional Diversification of Diiron-containing Enzymes

“…This implies the iron-bound oxidant occurs in fixed positions; thus, differences in catalytic activity are likely the result of differences in presentation of the substrate to the oxidant. This could occur via changes in the amino acid sequence of the enzyme close to the active site as was described for the oleate hydroxylase (27,34) or from differences in the nature of the substrate that could perhaps subtly alter the register of the fatty acid in the binding cavity (37). Support for changes in register influencing reaction outcome comes from analysis of mutants of the oleate desaturase capable of hydroxylation, in which the hydroxylation/desaturation ratio was 5-9-fold higher for 18-carbon versus 16-carbon substrates (27).…”

“…Support for changes in register influencing reaction outcome comes from analysis of mutants of the oleate desaturase capable of hydroxylation, in which the hydroxylation/desaturation ratio was 5-9-fold higher for 18-carbon versus 16-carbon substrates (27). The presence of functional groups in the acyl chain can be envisaged to affect the substratebinding mode as described above for the variant soluble desaturase (37).…”