Isolation of a Δ5 desaturase gene from Euglena gracilis and functional dissection of its HPGG and HDASH motifs.
Delta (Δ) 5 desaturase is a key enzyme for the biosynthesis of health-beneficial long chain polyunsaturated fatty acids such as arachidonic acid (ARA, C20:4n-6), eicosapentaenoic acid (C20:5n-3) and docosahexaenoic acid (C22:6n-3) via the "desaturation and elongation" pathways. A full length Δ5 desaturase gene from Euglena gracilis (EgΔ5D) was isolated by cloning the products of polymerase chain reaction with degenerate oligonucleotides as primers, followed by 5' and 3' rapid amplification of cDNA ends. The whole coding region of EgΔ5D was 1,350 nucleotides in length and encoded a polypeptide of 449 amino acids. BlastP search showed that EgΔ5D has about 39 % identity with a Δ5 desaturase of Phaeodactylum tricornutum. In a genetically modified dihomo-gamma-linoleic acid (DGLA, C20:3n-6) producing Yarrowia lipolytica strain, EgΔ5D had strong Δ5 desaturase activity with DGLA to ARA conversion of more than 24 %. Functional dissection of its HPGG and HDASH motifs demonstrated that both motifs were important, but not necessary in the exact form as encoded for the enzyme activity of EgΔ5D. A double mutant EgΔ5D-34G158G with altered sequences within both HPGG and HDASH motifs was generated and exhibited Δ5 desaturase activity similar to the wild type EgΔ5D. Codon optimization of the N-terminal region of EgΔ5D-34G158G and substitution of the arginine with serine at residue 347 improved substrate conversion to 27.6 %.
Biochemical Sciences and Engineering, Central Research and Development, E. I. du Pont de Nemours and Company, Wilmington, DE 19880, USA.
Dana
D
Walters Pollak
Michael W
MW
Bostick
Hyeryoung
H
Yoon
Jamie
J
Wang
Dieter H
DH
Hollerbach
Hongxian
H
He
Howard G
HG
Damude
Hongxiang
H
Zhang
Narendra S
NS
Yadav
Seung-Pyo
SP
Hong
Pamela
P
Sharpe
Zhixiong
Z
Xue
Quinn
Q
Zhu
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D005056:Euglena gracilis / Q000201:enzymology* / Q000235:genetics*; D044943:Fatty Acid Desaturases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D016415:Sequence Alignment
D044943:Fatty Acid Desaturases