Identification and functional characterization of the moss Physcomitrella patens delta5-desaturase gene involved in arachidonic and eicosapentaenoic acid biosynthesis.
The moss Physcomitrella patens contains high levels of arachidonic acid and lesser amounts of eicosapentaenoic acid. Here we report the identification and characterization of a delta5-desaturase from P. patens that is associated with the synthesis of these fatty acids. A full-length cDNA for this desaturase was identified by data base searches based on homology to sequences of known delta5-desaturase cDNAs from fungal and algal species. The resulting P. patens cDNA encodes a 480-amino acid polypeptide that contains a predicted N-terminal cytochrome b5-like domain as well as three histidine-rich domains. Expression of the enzyme in Saccharomyces cerevisiae resulted in the production of the delta5-containing fatty acid arachidonic acid in cells that were provided di-homo-gamma-linolenic acid. In addition, the expressed enzyme generated delta5-desaturation products with the C20 substrates omega-6 eicosadienoic and omega-3 eicosatrienoic acids, but no products were detected with the C18 fatty acid linoleic and alpha-linolenic acids or with the C22 fatty acid adrenic and docosapentaenoic acids. When the corresponding P. patens genomic sequence was disrupted by replacement through homologous recombination, a dramatic alteration in the fatty acid composition was observed, i.e. an increase in di-homo-gamma-linolenic and eicosatetraenoic acids accompanied by a concomitant disappearance of the delta5-fatty acid arachidonic and eicosapentaenoic acids. In addition, overexpression of the P. patens cDNA in protoplasts isolated from a disrupted line resulted in the restoration of arachidonic acid synthesis.
Kaewsuwan
Songsri
S
Department of Pharmacognosy, Mahidol University, 447 Sri-Ayudhya Road, Ratchathewi, Bangkok 10400, Thailand.
Cahoon
Edgar B
EB
United States Department of Agriculture Agricultural Research Service, Plant Genetics Research Unit, Donald Danforth Plant Science Center, St. Louis, Missouri 63132.
Perroud
Pierre-François
PF
Department of Biology, Washington University in St. Louis, S Louis, Missouri 63130-4899.
Wiwat
Chanpen
C
Department of Microbiology, Faculty of Pharmacy, Mahidol University, 447 Sri-Ayudhya Road, Ratchathewi, Bangkok 10400, Thailand.
Panvisavas
Nathinee
N
Department of Plant Sciences, Faculty of Sciences, Mahidol University, Rama VI Road, Bangkok 10400, Thailand.
Quatrano
Ralph S
RS
Department of Biology, Washington University in St. Louis, S Louis, Missouri 63130-4899. Electronic address: rsq@biology.wustl.edu.
Cove
David J
DJ
Department of Biology, Washington University in St. Louis, S Louis, Missouri 63130-4899.
Bunyapraphatsara
Nuntavan
N
Department of Pharmacognosy, Mahidol University, 447 Sri-Ayudhya Road, Ratchathewi, Bangkok 10400, Thailand. Electronic address: pynby@mahidol.ac.th.
D016718:Arachidonic Acid / Q000096:biosynthesis*; D044002:Bryophyta; D019068:Bryopsida / Q000201:enzymology*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D000090943:Delta-5 Fatty Acid Desaturase; D004537:Eicosanoic Acids; D015118:Eicosapentaenoic Acid / Q000096:biosynthesis*; D044943:Fatty Acid Desaturases / Q000235:genetics*; D010940:Plant Proteins / Q000235:genetics; D011523:Protoplasts / Q000378:metabolism
D018076:DNA, Complementary; D000090943:Delta-5 Fatty Acid Desaturase; D004537:Eicosanoic Acids; D010940:Plant Proteins; D016718:Arachidonic Acid; D015118:Eicosapentaenoic Acid; D044943:Fatty Acid Desaturases