title: 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.
authors.affiliation: Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Datun Road, Chaoyang District, Beijing, 100101, China, lysong@genetics.ac.cn.
authors.forename: Li-Ying
authors.initials: LY
authors.lastname: Song
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.forename: Shu-Fen
authors.initials: SF
authors.lastname: Li
authors.forename: Jun
authors.initials: J
authors.lastname: Hu
authors.forename: Wei-Bo
authors.initials: WB
authors.lastname: Yin
authors.forename: Yu-Hong
authors.initials: YH
authors.lastname: Chen
authors.forename: Shan-Ting
authors.initials: ST
authors.lastname: Hao
authors.forename: Bai-Lin
authors.initials: BL
authors.lastname: Wang
authors.forename: Richard R-C
authors.initials: RR
authors.lastname: Wang
authors.forename: Zan-Min
authors.initials: ZM
authors.lastname: Hu
mesh_terms: D000595:Amino Acid Sequence; D044943:Fatty Acid Desaturases / Q000235:genetics / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism*; D008958:Models, Molecular; D016296:Mutagenesis; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D005227:Fatty Acids; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D010088:Oxidoreductases; D044943:Fatty Acid Desaturases; C115532:sphingolipid desaturase