Dataset Information

Two clusters of residues contribute to the activity and substrate specificity of Fm1, a bifunctional oleate and linoleate desaturase of fungal origin.

ABSTRACT: Polyunsaturated fatty acids (PUFAs) have important industrial, physiological, and nutritional properties. Plants use the sequential activities of FAD2 and FAD3 desaturases to convert 18:1?9 to the important PUFA 18:3?9,12,15, whereas the fungus Fusarium verticillioides 7600 uses the bifunctional desaturase Fm1 for both reactions. Here, we used a combination of sequence comparisons, structural modeling, and mutagenesis experiments to investigate Fm1's regioselectivity and identified two functionally relevant clusters of residues that contribute to Fm1 activity. We found that cluster I (Leu153, Phe157, and His194), located near the catalytic iron ions, predominantly affects activity, whereas cluster II (Tyr280, His284, and Leu287), located in a helix forming the entrance to the substrate-binding pocket, mainly specifies 15-desaturation. Individual or combined substitutions of cluster II residues substantially reduced 15-desaturation. The combination of F157W from cluster I with Y280L, H284V, and L287T from cluster II created an increased-activity variant that almost completely lost the ability to desaturate at C15 and acted almost exclusively as a 12-desaturase. No variants were identified in which 15-desaturation occurred in the absence of 12-desaturation. Fm1 displayed only traces of activity with C16 substrate, but several cluster I variants exhibited increased activity with both 18:1 and 16:1 substrates, converting 16:1?9 to 16:3?9,12,15, consistent with Fm1 performing sequential v + 3 desaturation reactions at C12 and then C15. We propose that cluster II residues interact with the substrate headgroup when the acyl chain contains both ?9 and ?12 double bonds, in which case C15 becomes positioned adjacent to the di-iron site enabling a second v + 3 desaturation.

SUBMITTER: Cai Y

PROVIDER: S-EPMC6314118 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Two clusters of residues contribute to the activity and substrate specificity of Fm1, a bifunctional oleate and linoleate desaturase of fungal origin.

Cai Yuanheng Y Yu Xiao-Hong XH Liu Qun Q Liu Chang-Jun CJ Shanklin John J

The Journal of biological chemistry 20181022 51

Polyunsaturated fatty acids (PUFAs) have important industrial, physiological, and nutritional properties. Plants use the sequential activities of FAD2 and FAD3 desaturases to convert 18:1Δ9 to the important PUFA 18:3Δ9,12,15, whereas the fungus <i>Fusarium verticillioides</i> 7600 uses the bifunctional desaturase Fm1 for both reactions. Here, we used a combination of sequence comparisons, structural modeling, and mutagenesis experiments to investigate Fm1's regioselectivity and identified two fu ...[more]

PMID: 30348899

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Project description:BACKGROUND:Trypanosomes can synthesize polyunsaturated fatty acids. Previously, we have shown that they possess stearoyl-CoA desaturase (SCD) and oleate desaturase (OD) to convert stearate (C18) into oleate (C18:1) and linoleate (C18:2), respectively. Here we examine if OD is essential to these parasites. METHODOLOGY:Cultured procyclic (insect-stage) form (PCF) and bloodstream-form (BSF) Trypanosoma brucei cells were treated with 12- and 13-thiastearic acid (12-TS and 13-TS), inhibitors of OD, and the expression of the enzyme was knocked down by RNA interference. The phenotype of these cells was studied. PRINCIPAL FINDINGS:Growth of PCF T. brucei was totally inhibited by 100 µM of 12-TS and 13-TS, with EC(50) values of 40±2 and 30±2 µM, respectively. The BSF was more sensitive, with EC(50) values of 7±3 and 2±1 µM, respectively. This growth phenotype was due to the inhibitory effect of thiastearates on OD and, to a lesser extent, on SCD. The enzyme inhibition caused a drop in total unsaturated fatty-acid level of the cells, with a slight increase in oleate but a drastic decrease in linoleate level, most probably affecting membrane fluidity. After knocking down OD expression in PCF, the linoleate content was notably reduced, whereas that of oleate drastically increased, maintaining the total unsaturated fatty-acid level unchanged. Interestingly, the growth phenotype of the RNAi-induced cells was similar to that found for thiastearate-treated trypanosomes, with the former cells growing twofold slower than the latter ones, indicating that the linoleate content itself and not only fluidity could be essential for normal membrane functionality. A similar deleterious effect was found after RNAi in BSF, even with a mere 8% reduction of OD activity, indicating that its full activity is essential. CONCLUSIONS/SIGNIFICANCE:As OD is essential for trypanosomes and is not present in mammalian cells, it is a promising target for chemotherapy of African trypanosomiasis.

Project description: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 16:0Delta7 desaturase FAD5 (ADS3) was retargeted to the cytoplasm, regiospecificity shifted 70-fold, Delta7 to Delta9. Conversely, retargeting of two related cytoplasmic 16:0Delta9 Arabidopsis desaturases (ADS1 and ADS2) to the plastid, shifted regiospecificity approximately 25-fold, Delta9 to Delta7. All three desaturases exhibited Delta9 regiospecificity when expressed in yeast, with desaturated products found predominantly on phosphatidylcholine. Coexpression of each enzyme with cucumber monogalactosyldiacylglycerol (MGDG) synthase in yeast conferred Delta7 desaturation, with 16:1Delta7 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 Delta7 regiospecificity is thus attributable to plastidial retargeting of the enzyme by addition of a transit peptide to a cytoplasmic Delta9 desaturase rather than the numerous sequence differences within the catalytic portion of ADS enzymes. The MGDG-dependent desaturase activity enabled plants to synthesize 16:1Delta7 and its abundant metabolite, 16:3Delta(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.

Dataset Information

Two clusters of residues contribute to the activity and substrate specificity of Fm1, a bifunctional oleate and linoleate desaturase of fungal origin.

Publications

Two clusters of residues contribute to the activity and substrate specificity of Fm1, a bifunctional oleate and linoleate desaturase of fungal origin.

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