Distinct roles of endoplasmic reticulum cytochrome b5 and fused cytochrome b5-like domain for rat Delta6-desaturase activity.
The Delta6-desaturase catalyzes key steps in long-chain polyunsaturated fatty acid biosynthesis. Although the gene coding for this enzyme has been isolated in diverse animal species, the protein structure remains poorly characterized. In this work, rat Delta6-desaturase expressed in COS-7 cells was shown to localize in the endoplasmic reticulum. As the enzyme contains an N-terminal cytochrome b5-like domain, we investigated by site-directed mutagenesis the role of this domain in the enzyme activity. The typical HPGG motif of the cytochrome b5-like domain, and particularly histidine in this motif, is required for the activity of the enzyme, whatever the substrate. Neither endogenous COS-7 cytochrome b5 nor coexpressed rat endoplasmic reticulum cytochrome b5 could rescue the activity of mutated forms of Delta6-desaturase. Moreover, when rat endoplasmic reticulum cytochrome b5 was coexpressed with wild-type desaturase, both proteins interacted and Delta6-desaturase activity was significantly increased. The identified interaction between these proteins is not dependent on the desaturase HPGG motif. These data suggest distinct and essential roles for both the desaturase cytochrome b5-like domain and free endoplasmic reticulum cytochrome b5 for Delta6-desaturase activity.
Guillou
Hervé
H
Laboratoire de Biochimie, Institut National de la Recherche Agronomique-Ecole Nationale Supérieure Agronomique, Rennes, France.
D'Andrea
Sabine
S
Rioux
Vincent
V
Barnouin
Romain
R
Dalaine
Stéphanie
S
Pedrono
Frédérique
F
Jan
Sophie
S
Legrand
Philippe
P
D000818:Animals; D019556:COS Cells; D002849:Chromatography, Gas; D015786:Cytochromes b5 / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism; D005786:Gene Expression Regulation; D000882:Haplorhini; D007150:Immunohistochemistry; D009154:Mutation / Q000235:genetics; D017434:Protein Structure, Tertiary; D051381:Rats; D013230:Stearoyl-CoA Desaturase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D005227:Fatty Acids; D015786:Cytochromes b5; D013230:Stearoyl-CoA Desaturase; C474452:delta 6-desaturase, rat