Cloning of a novel omega-6 desaturase from flax (Linum usitatissimum L.) and its functional analysis in Saccharomyces cerevisiae.
The Delta12 desaturase represents a diverse gene family in plants and is responsible for conversion of oleic acid (18:1) to linoleic acid (18:2). Several members of this family are known from plants like Arabidopsis and Soybean. Using primers from conserved C- and N-terminal regions, we have cloned a novel Delta12 desaturase gene amplified from flax genomic DNA, denoted as LuFAD2-2. This intron-less gene is 1,149-base pair long encoding 382 amino acids-putative membrane-bound Delta12 desaturase protein. Sequence comparisons show that the novel sequence has 85% similarity with previously reported flax Delta12 desaturase at amino acid level and shows typical features of membrane-bound desaturase such as three conserved histidine boxes along with four membrane-spanning regions that are universally present among plant desaturases. The signature amino acid sequence 'YNNKL' was also found to be present at the N terminus of the protein, which is necessary and sufficient for ER localization of enzyme. Neighbor-Joining tree generated from the sequence alignment grouped LuFAD2-2 among the other FAD2 sequences from Ricinus, Hevea, Jatropha, and Vernicia. When LuFAD2-2 and LuFAD2 were expressed in Saccharomyces cerevisiae, they could convert the oleic acid to linoleic acid, with an average conversion rate of 5.25 and 8.85%, respectively. However, exogenously supplied linoleic acid was feebly converted to linolenic acid suggesting that LuFAD2-2 encodes a functional FAD2 enzyme and has substrate specificity similar to LuFAD2.
Khadake
Rupali M
RM
Bharati Vidyapeeth University, Pune, India.
Ranjekar
Prabhakar K
PK
Harsulkar
Abhay M
AM
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004789:Enzyme Activation; D004795:Enzyme Stability; D044943:Fatty Acid Desaturases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019597:Flax / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000502:physiology*
D011994:Recombinant Proteins; D044943:Fatty Acid Desaturases; C040448:delta-12 fatty acid desaturase