Switching desaturase enzyme specificity by alternate subcellular targeting

Abstract

The functionality, substrate specificity, and regiospecificity of enzymes typically evolve by the accumulation of mutations in the catalytic portion of the enzyme until new properties arise. However, emerging evidence suggests enzyme functionality can also be influenced by metabolic context. When the plastidial Arabidopsis 16:0⌬ 7 desaturase FAD5 (ADS3) was retargeted to the cytoplasm, regiospecificity shifted 70-fold, ⌬ 7 to ⌬ 9 . Conversely, retargeting of two related cytoplasmic 16:0⌬ 9 Arabidopsis desaturases (ADS1 and ADS2) to the plastid, shifted regiospecificity Ϸ25-fold, ⌬ 9 to ⌬ 7 . All three desaturases exhibited ⌬ 9 regiospecificity when expressed in yeast, with desaturated products found predominantly on phosphatidylcholine. Coexpression of each enzyme with cucumber monogalactosyldiacylglycerol (MGDG) synthase in yeast conferred ⌬ 7 desaturation, with 16:1⌬ 7 accumulating specifically on the plastidial lipid MGDG. Positional analysis is consistent with ADS desaturation of 16:0 on MGDG. The lipid headgroup acts as a molecular switch for desaturase regiospecificity. FAD5 ⌬ 7 regiospecificity is thus attributable to plastidial retargeting of the enzyme by addition of a transit peptide to a cytoplasmic ⌬ 9 desaturase rather than the numerous sequence differences within the catalytic portion of ADS enzymes. The MGDG-dependent desaturase activity enabled plants to synthesize 16:1⌬ 7 and its abundant metabolite, 16:3⌬ 7,10,13 . Bioinformatics analysis of the Arabidopsis genome identified 239 protein families that contain members predicted to reside in different subcellular compartments, suggesting alternative targeting is widespread. Alternative targeting of bifunctional or multifunctional enzymes can exploit eukaryotic subcellular organization to create metabolic diversity by permitting isozymes to interact with different substrates and thus create different products in alternate compartments.M etabolic diversity in living systems arises primarily from biotransformations catalyzed by enzymes; indeed life itself depends on the specificity of enzymes. The unique functionality, substrate specificity, and regiospecificity of an enzyme typically evolves by the gradual accumulation of changes in the catalytic portion of the enzyme until new properties arise. However, there is an emerging body of evidence suggesting that an enzyme's functional characteristics can also be affected by its metabolic context and that temporal and spatial dynamics of enzyme interactions can be important determinants of enzyme functionality (1). Examples include the ''rewiring'' of mitogen-activated protein kinase pathways on alternative scaffolds (2); the localization-dependent interactions of transcription factors with RNA polymerase in the nucleus (reviewed in ref. 3); the interchangeable tissue-specific roles of the transcription factors WER and GL1 in plant epidermal hair development (4); the modification of laccase͞peroxidase activity in the extracellular matrix by specific dirigent proteins (reviewed in ref. 5); the altered gati...

Maize cDNAs Expressed in Endosperm Encode Functional Farnesyl Diphosphate Synthase with Geranylgeranyl Diphosphate Synthase Activity

“…Metabolic context has been shown to affect enzyme regiospecificity when subcellular location was altered as in the case of a fatty acid desaturase (Heilmann et al, 2004). The distribution of isoprenoid synthase products in a given organism is also influenced by nonenzymatic factors such as salt and metal ion concentrations, or the available concentration of substrates, conditions that have been studied in vitro (Fujii, 1980;Matsuoka et al, 1991).…”

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).…”

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

“…The identification of a prime determinant of regioselectivity 27 Å from the diiron site, at which double-bond formation occurs, is perhaps surprising, however, that regioselectivity can be affected by interaction of the acyl carrier moiety and desaturase is not without precedent. Within the evolutionarily distinct class of integral membrane enzymes, the FAD5 desaturase was shown to perform Δ7 desaturation when presented with 16∶0 esterified to monogalactosyldiacylglycerol, but Δ9 desaturation when presented with 16∶0 esterified to phosphatidyl choline (16). Thus, for both soluble and integral membrane desaturase, regioselectivity is controlled remotely, albeit by different mechanisms.…”