Identification of the substrate recognition region in the [Delta]^sup 6^-fatty acid and [Delta]^sup 8^-sphingolipid desaturase by fusion mutagenesis

• Published in: Planta 2014-04, Vol.239 (4), p.753
• Main Authors: Song, Li-ying, Zhang, Yan, Li, Shu-fen, Hu, Jun, Yin, Wei-bo, Chen, Yu-hong, Hao, Shan-ting, Wang, Bai-lin, Wang, Richard R-c, Hu, Zan-min
• Format: Article
• Language: English
• Subjects: Amino acids; Fatty acids; Residues; Substrates; Yeasts
• ISSN: 0032-0935; 1432-2048
• DOI: 10.1007/s00425-013-2006-x

[Delta]^sup 8^-sphingolipid desaturase and [Delta]^sup 6^-fatty acid desaturase share high protein sequence identity. Thus, it has been hypothesized that [Delta]^sup 6^-fatty acid desaturase is derived from [Delta]^sup 8^-sphingolipid desaturase; however, there is no direct proof. The substrate recognition regions of [Delta]^sup 6^-fatty acid desaturase and [Delta]^sup 8^-sphingolipid desaturase, which aid in understanding the evolution of these two enzymes, have not been reported. A blackcurrant [Delta]^sup 6^-fatty acid desaturase and a [Delta]^sup 8^-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 [Delta]^sup 6^- and [Delta]^sup 8^-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 [Delta]^sup 6^-substrate recognition, and the last two regions were crucial for [Delta]^sup 8^-substrate recognition; and (3) amino acid residues 114-276 of [Delta]^sup 6^-fatty acid desaturase contained the substrate recognition site(s) responsible for discrimination between ceramide (a substrate of [Delta]^sup 8^-sphingolipid desaturase) and acyl-PC (a substrate of [Delta]^sup 6^-fatty acid desaturase). Substituting the amino acid residues 114-276 of RnD8A with those of RnD6C resulted in a gain of [Delta]^sup 6^-desaturase activity in the fusion protein but a loss in [Delta]^sup 8^-sphingolipid desaturase activity. In conclusion, several regions important for the substrate recognition of [Delta]8-sphingolipid desaturase and [Delta]^sup 6^-fatty acid desaturase were identified, which provide clues in understanding the relationship between the structure and function in desaturases.[PUBLICATION ABSTRACT]