Characterization and molecular docking of new Δ17 fatty acid desaturase genes from Rhizophagus irregularis and Octopus bimaculoides

Abstract

Fatty acid desaturases are key enzymes in the biosynthesis of n-3 polyunsaturated fatty acids (PUFAs) via conversion of n-6 polyunsaturates to their n-3 counterparts. In this study, we reported the characterization and molecular docking of Δ17 desaturases from Rhizophagus irregularis and Octopus bimaculoides. These two new desaturase genes were screened using the known Δ17 desaturase gene (oPaFADS17) from Pythium aphanidermatum as a template. Analysis of their genes revealed that the sequences of oRiFADS17 and oObFADS17 contained the typical His-rich motifs (one HXXXH and two HXXHH). They were then expressed in Saccharomyces cerevisiae INVSc1 to examine their activities and substrate preferences. Our results show that the two candidate n-3 desaturases possess a strong Δ17 desaturase activity, exhibiting remarkable increase in desaturation activity on C20 fatty acids compared to C18 fatty acids. To the best of our knowledge, oRiFADS17 desaturase has greater (3-4 fold) catalytic activity for C18 substrates than other reported Δ17 desaturases and oObFADS17 is the first reported Δ17 desaturase in sea mollusks. Characterization of these two new desaturases will be of greater value for genetic engineering in industrial production of eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3). Due to lack of crystal structure information about n-3 desaturases, for the first time, the view of their predicted structures, binding pockets and substrate tunnels was clearly observed based on molecular docking. This will contribute to strengthening our understanding of the structure-function relationships of n-3 fatty acid desaturases.

Fig. 1. Schematic diagram of the fatty acid pathways for EPA and DHA synthesis.

Fig. 2. Multiple sequence alignment of 16 reported n-3 fatty acid desaturases and 4 hypothetical desaturases.

Fig. 3. Phylogenetic analysis showing the relationship among 16 selected desaturases and 4 hypothetical proteins.

Fig. 4. Construction and verification of INVSc1 plasmids. (a) Construction of INVSc1 plasmids pYES2-oAcFADS17, pYES2-oBgFADS17, pYES2-oObFADS17, pYES2-oRiFADS17. (b) Verification of INVSc1 transformants. M: maker; lane 1: control; lanes 2-5: pYES2-oAcFADS17, pYES2-oBgFADS17, pYES2-oObFADS17, pYES2-oRiFADS17.

Fig. 5. Verification of translation levels of yeast transformants. (a) Western blotting of recombinant proteins expressed in S. cerevisiae. M: marker; C: control protein oPaFADS17; arrows in the western blotting indicated the location of the target protein. (b) Western-blot gray scale result showed that the average translation levels of 4 hypothetical Δ17 desaturases were almost the same amount.

Fig. 6. Conversion rates of yeast transformants on different fatty acids. (a) Rate of substrate conversion of oPaFADS17, oAcFADS17, oBgFADS17, oObFADS17 and oRiFADS17 in S. cerevisiae. (b) Rate of substrate conversion of sdd17, Pi-D17, Pr-D17, Ps-D17, fat-1, oPaFADS17, oRiFADS17 and oObFADS17.

Fig. 7. Homology modeling of three-dimensional structures and molecular docking of oObFADS17 and oRiFADS17.