Chimeras of Delta6-fatty acid and Delta8-sphingolipid desaturases.
The Borago officinalis Delta6 fatty acid desaturase (Boofd6) shares 58% identity in its amino acid sequence with Boofd8, a Delta8 sphingolipid desaturase from the same plant species. In order to localise the distinct catalytic properties of Boofd6 and Boofd8 to individual regions within them, a set of chimeras of these two enzymes were constructed and expressed in yeast. Chimera 2 is different from the other chimeras and Boofd6 in that it did not have any detectable desaturase activity on 18 carbon fatty acids. However, it desaturated C16 palmitoleic and C14 myristoleic acid, and the conversion rate for the later one was more than three times higher than that of Boofd6. These results suggest that the predicted membrane helices 1 and 2 of Boofd6 are involved in forming the substrate-binding site. This site appears to place constraints on the chain length of fatty acid substrates, which is similar to hydrophobic substrate binding pockets.
Libisch
B
B
Department of Agricultural Sciences, IACR-Long Ashton Research Station, Bristol, United Kingdom.
Michaelson
L V
LV
Lewis
M J
MJ
Shewry
P R
PR
Napier
J A
JA
D044943:Fatty Acid Desaturases / Q000235:genetics / Q000378:metabolism*; D050602:Linoleoyl-CoA Desaturase; D019684:Magnoliopsida / Q000201:enzymology*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D013379:Substrate Specificity
D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D010088:Oxidoreductases; D044943:Fatty Acid Desaturases; D050602:Linoleoyl-CoA Desaturase; C115532:sphingolipid desaturase