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

Δ-sphingolipid desaturase and Δ-fatty acid desaturase share high protein sequence identity. Thus, it has been hypothesized that Δ-fatty acid desaturase is derived from Δ-sphingolipid desaturase; however, there is no direct proof. The substrate recognition regions of Δ-fatty acid desaturase and Δ-sphingolipid desaturase, which aid in understanding the evolution of these two enzymes, have not been reported. A blackcurrant Δ-fatty acid desaturase and a Δ-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 Δ- and Δ-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 Δ-substrate recognition, and the last two regions were crucial for Δ-substrate recognition; and (3) amino acid residues 114-276 of Δ-fatty acid desaturase contained the substrate recognition site(s) responsible for discrimination between ceramide (a substrate of Δ-sphingolipid desaturase) and acyl-PC (a substrate of Δ-fatty acid desaturase). Substituting the amino acid residues 114-276 of RnD8A with those of RnD6C resulted in a gain of Δ-desaturase activity in the fusion protein but a loss in Δ-sphingolipid desaturase activity. In conclusion, several regions important for the substrate recognition of Δ8-sphingolipid desaturase and Δ-fatty acid desaturase were identified, which provide clues in understanding the relationship between the structure and function in desaturases. [ABSTRACT FROM AUTHOR]