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Catalytic Plasticity of Fatty Acid Modification Enzymes Underlying Chemical Diversity of Plant Lipids

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Science  13 Nov 1998:
Vol. 282, Issue 5392, pp. 1315-1317
DOI: 10.1126/science.282.5392.1315

Abstract

Higher plants exhibit extensive diversity in the composition of seed storage fatty acids. This is largely due to the presence of various combinations of double or triple bonds and hydroxyl or epoxy groups, which are synthesized by a family of structurally similar enzymes. As few as four amino acid substitutions can convert an oleate 12-desaturase to a hydroxylase and as few as six result in conversion of a hydroxylase to a desaturase. These results illustrate how catalytic plasticity of these diiron enzymes has contributed to the evolution of the chemical diversity found in higher plants.

All higher plants contain one or more oleate desaturases that catalyze the O2-dependent insertion of a double bond between carbons 12 and 13 of lipid-linked oleic acid (18:1Δ9) to produce linoleic acid (18:2Δ9,12) (1). In contrast, only 14 species in 10 plant families have been found to accumulate the structurally related hydroxy fatty acid, ricinoleic acid (d-12-hydroxyoctadec-cis-9-enoic acid) (2), which is synthesized by an oleate hydroxylase that exhibits a high degree of sequence similarity to oleate desaturases (3). The oleate desaturases and hydroxylases are integral membrane proteins, which are members of a large family of functionally diverse enzymes that includes alkane hydroxylase, xylene monooxygenase, carotene ketolase, and sterol methyloxidase (1). These nonheme iron–containing enzymes use a diiron cluster for catalysis (4) and contain three equivalent histidine clusters that have been implicated in iron binding and shown to be essential for catalysis (1). This class of proteins exhibits no significant sequence identity to the soluble diiron-containing enzymes which represent a similar diversity of enzymatic activities that include plant acyl-ACP desaturases, methane monooxygenase, propene monooxygenase, and the R2 component of ribonucleotide reductase (1, 5). The catalytic activities of these enzymes has been mimicked by a synthetic diiron-containing complex with a coordination sphere composed entirely of nitrogen ligands (6).

The oleate hydroxylase from the crucifer Lesquerella fendleri has about 81% sequence identity to the oleate desaturase from the crucifer Arabidopsis thaliana and about 71% sequence identity to the oleate hydroxylase from Ricinus communis (7). The observation that these crucifer desaturase and hydroxylase enzymes are more similar than the two hydroxylases, and the presence of ricinoleic acid in a small number of distantly related plant species, suggests that the capacity to synthesize ricinoleate has arisen independently several times during the evolution of higher plants, by the genetic conversion of desaturases to hydroxylases.

Comparison of the amino acid sequences of the hydroxylases fromL. fendleri and R. communis with the sequences for oleate desaturases from Arabidopsis, Zea mays, Glycine max (two sequences), R. communis, and Brassica napus revealed that only seven residues were strictly conserved in all of the six desaturases but divergent in both of the hydroxylases. The role of these seven residues was assessed by using site-directed mutagenesis to replace the residues found in the Lesquerella hydroxylase, LFAH12, with those from the equivalent positions in the desaturases (8, 9). In a reciprocal experiment, we replaced the seven residues in theArabidopsis FAD2 oleate desaturase with the correspondingLesquerella hydroxylase residues (10). The activity of the modified and unmodified genes was then determined by expressing them in yeast and transgenic plants before analyzing the composition of the total fatty acids. Technical difficulties limited the utility of direct measurements of enzyme activity in cell extracts (11).

The mutant hydroxylase and desaturase genes containing all seven substitutions (designated m7LFAH12 and m7FAD2, respectively) were expressed in yeast cells under transcriptional control of the GAL1 promoter. Transgenic cells were harvested after induction and their total fatty acid composition determined by gas chromatography. Wild-type yeast cells do not accumulate detectable concentrations of diunsaturated or hydroxylated fatty acids (12). Expression of FAD2 caused the accumulation of about 4% diunsaturated fatty acids (16:2 and 18:2) but no detectable hydroxy fatty acids (Fig. 1). Expression of LFAH12 caused the accumulation of about 1.4% diunsaturated fatty acids and 1.5% ricinoleic acid, confirming the mixed function of this enzyme (7). Cells expressing m7FAD2 accumulated ricinoleic acid to ∼0.5% of total fatty acids and had ∼50% reduction in the accumulation of diunsaturated fatty acids (Fig. 1). Thus, replacement of the seven residues (10) converted a strict desaturase to a bifunctional desaturase-hydroxylase comparable in activity to the unmodified Lesquerella hydroxylase.

Figure 1

Fatty acid composition of yeast cells expressing desaturase and hydroxylase genes. Cultures were induced in growth medium containing galactose, ∼2 × 108 cells were harvested, and fatty acids were extracted and modified for analysis by gas chromatography, as described (7). Values are the averages (±SE) obtained from five cultures of independent transformants.

The amount of desaturase activity of the LFAH12 enzyme is relatively low compared with its hydroxylase activity (7). However, yeast cells expressing LFAH12 accumulated linoleic and ricinoleic acids to similar concentrations, possibly because linoleic acid is more stable than ricinoleic acid in yeast cells. In cells expressing m7FAH12, the ratio of 18:2 diunsaturated fatty acid to ricinoleic acid was, on average, 43 times that in cells expressing LFAH12. There was also a 16-fold increase in the ratio of 16:2 diunsaturated fatty acid to ricinoleic acid. Notwithstanding the quantitative limitations of the assay system, noted above, these results indicate a major increase in desaturase activity and a decrease in hydroxylase activity upon introduction of the seven desaturase-equivalent residues into LFAH12.

The activity of the mutant enzymes in planta was examined by using the corresponding genes to produce stable transgenic plants in an Arabidopsis fad2 mutant, which is deficient in oleate desaturase activity (13). Expression of LFAH12 under transcriptional control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter resulted in accumulation of high concentrations of hydroxy fatty acids in seeds (7), but no detectable suppression of the fad2 mutant phenotype in leaves (Fig. 2). In contrast, expression of m7LFAH12 under the same circumstances resulted in complete suppression of the fad2 phenotype in 8 out of 10 transgenic plants analyzed (Fig. 2). There was an average 21-fold increase in the ratio of linoleate to oleate in leaf fatty acids and a small increase in the amount of linolenic acid. These results, which are consistent with the results of the yeast assays, confirm that expression of m7LFAH12 in plants deficient in oleate desaturation has identical phenotypic consequences to expressing a wild-type desaturase such as FAD2 (13).

Figure 2

Genetic complementation of theArabidopsis fad2 mutation with the m7LFAH12 gene. Measurements were made of the fatty acid composition of leaf lipids from wild-type, the fad2 mutant, and transgenicfad2 plants expressing LFAH12 or m7LFAH12, under the control of the CaMV 35S promoter. Values are means ± SE (n = 3).

To evaluate the effect of the seven mutations on the activity of the gene encoding FAD2, we expressed FAD2 and m7FAD2 in theArabidopsis fad2 mutant under the control of the strong seed-specific promoter from the B. rapa napin gene. As expected from previous studies (7), none of the 15 transgenic lines expressing the FAD2 gene accumulated detectable hydroxy fatty acids, although the ratio of linoleate to oleate accumulation was increased an average of 10-fold as compared with untransformed controls. In the transgenic lines expressing m7FAD2, the amount of hydroxylated fatty acids, which included ricinoleic, densipolic, and lesquerolic acids, composed up to 9.4% of total seed fatty acids (Fig. 3). The ratio of seed linoleate to oleate contents was increased an average of 6.4-fold (14), which indicated that m7FAD2 exhibited significant desaturase activity, albeit less than the wild-type FAD2 gene. The high concentrations of hydroxy fatty acid accumulation observed in transgenic plants expressing m7FAD2 indicated that the modified desaturase had comparable levels of hydroxylase activity, in the in planta assay, to the native Lesquerella hydroxylase enzyme.

Figure 3

Fatty acid content of seed lipids from independent transgenic Arabidopsis lines expressing m7FAD2 or m4FAD2 under control of the B. napus napin promoter. Abbreviations: ricinoleic acid (18:1-OH), densipolic acid (18:2-OH), and lesquerolic acid (20:1-OH).

To determine whether any single amino acid residue of the seven had a major effect on the ratio of hydroxylase to desaturase activities, we introduced each of the seven FAD2-equivalent residues (8) individually into the LFAH12 enzyme. None of the enzymes containing single amino acid substitutions had activities that differed significantly from the wild-type hydroxylase enzyme when expressed in yeast (14). We also tested seven modified LFAH12 genes containing all combinations of six desaturase-equivalent residues (Fig. 4). Each of the seven constructs produced a ratio of diunsaturated to hydroxylated fatty acids that was similar to the ratio produced by the m7FAH12 enzyme. Thus, as few as six residues principally determine the ratio of desaturation or hydroxylation activity. All lines showed somewhat reduced levels of desaturase activity, with the largest reductions of ∼40% seen in F218Y and G105A. Therefore, we made a construct in which both these changes were combined (xF218Y/G105A). This construct exhibited similar activity to the individual F218Y and G105A mutants (14), suggesting that their effects are redundant and that the observed changes in activity result from interactions of more than two of the seven residues. Considered together, these results indicate that no single amino acid position plays an essential role in catalytic outcome. Rather, changes in activity result from a combined effect of several amino acid positions that have partially overlapping effects.

Figure 4

Contribution of individual amino acid substitutions to the activity of the modified Lesquerellahydroxylase. Seven derivatives of the m7LFAH12 gene containing all combinations of six out of seven substitutions were introduced into yeast cells, and the fatty acid composition of five independent cultures was measured. The “X” designation refers to the unmodified amino acid (that is, enzyme XI325M contains all of the seven substitutions except I325M).

Because four of the seven amino acids are adjacent to histidine residues that have been identified as essential to catalysis (1), we hypothesized that these four residues may be of greatest importance to the outcome of the reaction. A modified FAD2 enzyme, designated m4FAD2, was constructed in which these four amino acids were replaced by their equivalents from theLesquerella hydroxylase (T148N, A296V, S322A, M324I). Expression of m4FAD2 in seeds of wild-typeArabidopsis resulted in the accumulation of average concentrations of hydroxy fatty acids that were similar to those obtained with m7FAD2 (Fig. 3). Thus, only four changes are required to convert a strict desaturase to an enzyme that retains some desaturase activity but is also an efficient hydroxylase.

Biochemical and structural similarities between the desaturase and hydroxylase, in addition to recent kinetic isotope experiments, suggest that there is a common initial oxidation event at C-12 for both enzymes (15). Thus, it seems likely that the different functional outcomes represent a partitioning between two reaction pathways that diverge after initial C-12 hydrogen abstraction such that one pathway favors a second hydrogen abstraction whereas the other favors oxygen transfer. We envision that because no specific single amino acid change is required, and in view of the substantial effect of the four residues that abut the active site histidines, the differences between desaturase and hydroxylase outcome is influenced by changes in active site geometry. Examples of such changes might include the relative positioning of the substrate with respect to the iron center, the coordination geometry of the iron ions, or the active site hydrogen bonding network. Whatever the case, this mode of evolving new catalytic activity differs from the more general case in which the evolution of new activities involves the incorporation of new catalytic groups into the active site (16).

Acetylenic and epoxy fatty acids are produced by desaturation and epoxidation of double bonds by enzymes that are structurally similar to the enzymes described here (17). Thus, variations of the same catalytic center can catalyze the formation of at least four different functional groups in fatty acids. Because various combinations of these four functional groups define most of the chemical complexity found among the hundreds of different fatty acids that occur in higher plants (2), it is now apparent that most of the chemical complexity of plant fatty acids can be accounted for by divergence of a small number of desaturases. Extrapolating from the results described here, it also seems very likely that a small number of amino acid substitutions will account for the functional divergence of desaturases, hydroxylases, expoxgenases, and acetylenic bond–forming enzymes.

  • * These authors contributed equally to this work.

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