Desaturation and Hydroxylation

Abstract

Exchanging the identity of amino acids at four key locations within the Arabidopsis thaliana oleate desaturase (FAD2) and the Lesquerella fendleri hydroxylase/ desaturase (LFAH) was shown to influence partitioning between desaturation and hydroxylation (Broun, P., Shanklin, J., Whittle, E., and Somerville, C. (1998) Science 282, 1315-1317). We report that four analogous substitutions in the FAD2 sequence by their equivalents from the castor oleate hydroxylase result in hydroxy fatty acid accumulation in A. thaliana to the same levels as for the wild-type castor hydroxylase. We also describe the relative contribution of these substitutions, both individually and in combination, by analyzing the products resulting from their expression in A. thaliana and/or Saccharomyces cerevisiae. Yeast expression showed that M324V, a change reachable by a single point mutation, altered the product distribution ϳ49-fold, and that residue 148 is also a predominant determinant of reaction outcome. Comparison of residues at position 148 of FAD2, LFAH, and the Ricinus oleate hydroxylase prompted us to rationally engineer LFAH-N149I, a variant with ϳ1.9-fold increase in hydroxylation specificity compared with that of wild-type LFAH. Control experiments showed that the wild-type Arabidopsis thaliana FAD2 desaturase has inherent, low level, hydroxylation activity. Further, fatty acid desaturases from different kingdoms and with different regiospecificities exhibit similar intrinsic hydroxylase activity, underscoring fundamental mechanistic similarities between desaturation and hydroxylation. For LFAH mutants the hydroxylation:desaturation ratio is 5-9-fold higher for 18-carbon versus 16-carbon substrates, supporting our hypothesis that substrate positioning in the active site plays a key role in the partitioning of catalytic specificity.Hydroxy fatty acids are unusual fatty acids that are incorporated into seed triacylglycerols in several species of plants, the best characterized being Ricinus communis (castor) and Lesquerella fendleri (1, 2). In both of these plants, an oleate hydroxylase enzyme catalyzes the hydroxylation chemistry that converts oleate (cis-9-octadecenoic acid, or 18:1⌬ 9 ) to rici- (1), and Lesquerella, LFAH (2), are closely related to the common plant oleate desaturase enzyme (FAD2), which converts oleate (18:1 ⌬ 9 ) into linoleate (18:2⌬ 9,12 ). Indeed, LFAH actually retains both hydroxylase and desaturase activity, indicating that these two oxidation reactions can be catalyzed by the same enzyme. Amino acid sequence alignments (Table I) of these two oleate hydroxylases with several oleate desaturases indicated that there are only a few conserved desaturase residues that are not conserved in the hydroxylases (3). These seven residues (Arabidopsis thaliana FAD2 residues 63, 104, 148, 217, 295, 322, and 324) 3 were replaced with the corresponding residues of LFAH, and the resulting enzyme (designated m7FAD2 in Ref.3) was found to be sufficient to convert the desaturase into a bifunctional desaturase/hydroxylase. ...

Switching desaturase enzyme specificity by alternate subcellular targeting

“…Changes to an enzyme's regiospecificity typically require between two and six specific changes at key locations along the amino acid chain that occur over many generations (35,36). To accumulate mutations at these key sites, many additional mutations also accumulate, which tend to degrade attributes such as stability and turnover of the enzyme (37).…”

Identification of bifunctional Δ12/ω3 fatty acid desaturases for improving the ratio of ω3 to ω6 fatty acids in microbes and plants

“…Although it has previously been shown that desaturases can have additional functionality, such as acting as conjugases (25) or hydroxylases (26), different positional specificities depending on the type of lipid to which the substrate is conjugated (27), the chain length of the acyl group (28), or the ability of a soluble desaturase enzyme to catalyze two double bonds (⌬4 and ⌬9) into a saturated fatty acyl-ACP substrate (29), the ability of a desaturase enzyme to catalyze two double bonds (⌬12 and 3) on the same acyl chain (C18) has not previously been reported. It will be interesting to study how these ⌬12͞ 3 enzymes exert their dual positional specificity, because the regioselectivity of ⌬12 and 3 desaturases is Јv ϩ 3Ј (i.e., three carbons away from an existing double bond) and Ϫ3 (i.e., three carbons from the methyl end), respectively (30,31).…”

Domain Swapping Localizes the Structural Determinants of Regioselectivity in Membrane-bound Fatty Acid Desaturases of Caenorhabditis elegans

“…Eight highly conserved histidine residues have been shown to be required for function with the exception of one that is replaced by glutamine in some "front-end" desaturases, which introduce double bonds near C-1 in the substrate (3,31). Shanklin and colleagues (32) have identified a number of individual amino acids that are important for the chemoselectivity of the FAD2-like enzymes, which tend to have both desaturase and hydroxylase activities to varying degrees. A domain swapping study was undertaken by Napier and colleagues (33) to investigate the regioselectivity and substrate specificity of two front-end desaturases.…”