Identification of the substrate recognition region in the Δ6-fatty acid and Δ8-sphingolipid desaturase by fusion mutagenesis

Abstract

Δ⁸-sphingolipid desaturase and Δ⁶-fatty acid desaturase share high protein sequence identity. Thus, it has been hypothesized that Δ⁶-fatty acid desaturase is derived from Δ⁸-sphingolipid desaturase; however, there is no direct proof. The substrate recognition regions of Δ⁶-fatty acid desaturase and Δ⁸-sphingolipid desaturase, which aid in understanding the evolution of these two enzymes, have not been reported. A blackcurrant Δ⁶-fatty acid desaturase and a Δ⁸-sphingolipid desaturase gene, RnD6C and RnD8A, respectively, share more than 80 % identity in their coding protein sequences. In this study, a set of fusion genes of RnD6C and RnD8A were constructed and expressed in yeast. The Δ⁶- and Δ⁸-desaturase activities of the fusion proteins were characterized. Our results indicated that (1) the exchange of the C-terminal 172 amino acid residues can lead to a significant decrease in both desaturase activities; (2) amino acid residues 114-174, 206-257, and 258-276 played important roles in Δ⁶-substrate recognition, and the last two regions were crucial for Δ⁸-substrate recognition; and (3) amino acid residues 114-276 of Δ⁶-fatty acid desaturase contained the substrate recognition site(s) responsible for discrimination between ceramide (a substrate of Δ⁸-sphingolipid desaturase) and acyl-PC (a substrate of Δ⁶-fatty acid desaturase). Substituting the amino acid residues 114-276 of RnD8A with those of RnD6C resulted in a gain of Δ⁶-desaturase activity in the fusion protein but a loss in Δ⁸-sphingolipid desaturase activity. In conclusion, several regions important for the substrate recognition of Δ⁸-sphingolipid desaturase and Δ⁶-fatty acid desaturase were identified, which provide clues in understanding the relationship between the structure and function in desaturases.

Consensus mutagenesis and ancestral reconstruction provide insight into the substrate specificity and evolution of the front-end Δ6-desaturase family

“…Mutagenesis studies have revealed that the second putative TMH, the regions adjacent to the conserved histidine motifs and the Cterminus affect the regioselectivity and the preferred substrate chain length of membrane desaturases. 10,11,25,26 However, although Δ6-desaturases have been identified that naturally prefer either ω3 or ω6fatty acids, 23,[27][28][29][30][31] detailed mutagenesis studies to modify ω3/ω6fatty acid preference have not been performed, to the best of our knowledge. Similarly, the evolution of ω3/ω6fatty acid specificity, including divergence between MpΔ6des and OtΔ6des, has not been investigated in detail.…”

Plastidic Δ6 fatty-acid desaturases with distinctive substrate specificity regulate the pool of c18-pufas in the ancestral picoalgaostreococcus tauri

“…DES specificity relies on intricate substrate features, including the acyl-chain position, length, unsaturation features, and the acyl-carrier nature (Heilmann et al ., 2004a; Li, D et al ., 2016). Domain swapping between DES of distinctive specificity, together with further amino acid mutation, could highlight the primary importance of His-Box and surrounding region for front-end desaturase activity and substrate specificity (Song et al ., 2014; Li, D et al ., 2016; Watanabe et al ., 2016). However, neither the exact molecular features underlying DES (regio)specificity nor the hierarchical importance of the substrate features are yet clearly identified.…”

Identification of amino acid residues that determine the substrate specificity of mammalian membrane-bound front-end fatty acid desaturases

“…Protein engineering has been applied to understand the structure-function relationship. For instance, domain swapping has been used to identify the regioselective sites of nematode ⌬ 12 and 3 desaturases ( 12 ), a region determining the substrate specifi city of Aspergillus nidulans ⌬ 12 and 3 desaturases ( 13 ), and a substrate recognition region of black currant ⌬ 6 fatty acid desaturase and ⌬ 8 sphingolipid desaturase ( 14 ). Site-directed mutagenesis based on amino acid sequence comparison has been used to identify amino acids participating in the substrate specifi city of Mucor rouxii D6d ( 15 ), Siganus canaliculatus ⌬ 4 and D5d/D6d ( 16 ), and marine copepod ⌬ 9 desaturase ( 17 ).…”

“…The corresponding genes are positioned in a head-to-head confi guration on the rat genome, suggesting a paralogous relationship ( 11 ). Although their primary structures are highly homologous, they are in charge of mutually exclusive substrates: D6d catalyzes the conversion of linoleic acid (LA; 18:2 ⌬ 9,12) and ␣ -linolenic acid (18:3 ⌬ 9,12,15) into ␥ -linolenic acid (GLA; 18:3 ⌬ 6,9,12) and stearidonic acid (18:4 ⌬ 6,9,12,15), respectively, whereas D5d acts on dihomo-␥ -linolenic acid (DGLA; 20:3 ⌬ 8, 11,14) and eicosatetraenoic acid (20:4 ⌬ 8,11,14,17) to generate arachidonic acid (ARA; 20:4 ⌬ 5, 8,11,14) and eicosapentaenoic acid (20:5 ⌬ 5,8,11,14,17), respectively. To identify and evaluate the amino acid residues important for substrate selection of D6d, we performed additional analyses on the basis of the primary sequence of zebra fi sh bifunctional ⌬ 5/6 desaturase [zD5/6d ( 23 )] and the recently reported crystal structure of human stearoyl-CoA ( ⌬ 9) desaturase ( 24,25 ).…”