Biosynthesis of docosahexaenoic acid in Euglena gracilis:: Biochemical and molecular evidence for the involvement of a Δ4-fatty acyl group desaturase

被引:68
作者
Meyer, A [1 ]
Cirpus, P [1 ]
Ott, C [1 ]
Schlecker, R [1 ]
Zähringer, U [1 ]
Heinz, E [1 ]
机构
[1] Univ Hamburg, Inst Allgemeine Bot, D-22609 Hamburg, Germany
关键词
D O I
10.1021/bi034731y
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Docosahexaenoic acid (DHA) can be synthesized via alternative routes from which only the omega3/omega6-pathways involve the action of a Delta4-fatty acid desaturase. We examined the suitability of Euglena gracilis, Thraustochytrium sp., Schizochytrium sp., and Crypthecodinium cohnii to serve as sources for cloning a cDNA encoding a Delta4-fatty acid desaturase. For this purpose we carried Out in vivo labeling studies with radiolabeled C22 polyunsaturated fatty acid substrates. Schizochytrium sp. was unable to convert exogenously supplied [2-C-14]-docosapentaenoic acid (DPA, 22:5(Delta7,10,13,16,19)) to DHA, while E. gracilis and Thraustochytrium sp. carried out this desaturation very efficiently. Hydrogenation and cc-oxidation of the labeled DHA isolated from these two organisms showed that it was the result of direct Delta4-desaturation and not of substrate breakdown and resynthesis. To clone the desaturase gene, a cDNA library of E. gracilis was subjected to mass sequencing. A full-length clone with highest homology to the Delta4-desaturase of Thraustochytrium sp. was isolated, and its function was verified by heterologous expression in yeast. The desaturase efficiently converted DPA to DHA. Analysis of the substrate specificity demonstrated that the enzyme activity was not limited to C22 fatty acids, since it also efficiently desaturated C16 fatty acids. The enzyme showed strict Delta4-regioselectivity and required the presence of a AT-double bond in the substrate. Positional analysis of phosphatidylcholine revealed that the proportion of the Delta4-desaturated products was up to 20 times higher in the sn-2 position than in the sn-1 position.
引用
收藏
页码:9779 / 9788
页数:10
相关论文
共 55 条
[1]  
Abbadi A, 2001, EUR J LIPID SCI TECH, V103, P106, DOI 10.1002/1438-9312(200102)103:2<106::AID-EJLT106>3.0.CO
[2]  
2-V
[3]   Structure and regulation of the omega-3 polyunsaturated fatty acid synthase genes from the deep-sea bacterium Photobacterium profundum strain SS9 [J].
Allen, EE ;
Bartlett, DH .
MICROBIOLOGY-SGM, 2002, 148 :1903-1913
[4]   OPTIMIZATION OF PRODUCTION OF DOCOSAHEXAENOIC ACID (DHA) BY THRAUSTOCHYTRIUM-AUREUM ATCC 34304 [J].
BAJPAI, PK ;
BAJPAI, P ;
WARD, OP .
JOURNAL OF THE AMERICAN OIL CHEMISTS SOCIETY, 1991, 68 (07) :509-514
[5]   Heterologous reconstitution in yeast of the polyunsaturated fatty acid biosynthetic pathway [J].
Beaudoin, F ;
Michaelson, LV ;
Hey, SJ ;
Lewis, MJ ;
Shewry, PR ;
Sayanova, O ;
Napier, JA .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2000, 97 (12) :6421-6426
[6]  
BLIGH EG, 1959, CAN J BIOCHEM PHYS, V37, P911
[7]   POLYGLYCEROPHOSPHOLIPIDS OF EUGLENA-GRACILIS [J].
CALVAYRAC, R ;
DOUCE, R .
FEBS LETTERS, 1970, 7 (03) :259-+
[8]   Cloning, expression, and fatty acid regulation of the human Δ-5 desaturase [J].
Cho, HP ;
Nakamura, M ;
Clarke, SD .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1999, 274 (52) :37335-37339
[9]   Gas chromatography mass spectrometry methods for structural analysis of fatty acids [J].
Christie, WW .
LIPIDS, 1998, 33 (04) :343-353
[10]   The same rat Δ6-desaturase not only acts on 18- but also on 24-carbon fatty acids in very-long-chain polyunsaturated fatty acid biosynthesis [J].
D'Andrea, S ;
Guillou, H ;
Jan, S ;
Catheline, D ;
Thibault, JN ;
Bouriel, M ;
Rioux, V ;
Legrand, P .
BIOCHEMICAL JOURNAL, 2002, 364 (01) :49-55