Specific protein regions influence substrate specificity and product length in polyunsaturated fatty acid condensing enzymes.
We describe a condensing enzyme from Pythium irregulare (PirELO) that shows highest activity on the 18-carbon, Delta-6 desaturated fatty acids, stearidonic acid and gamma-linolenic acid. However, this enzyme is also capable of elongating a number of other fatty acids including the 20-carbon, Delta-5 desaturated fatty acid eicosapentaenoic acid. Surprisingly, a Phytophthora infestans condensing enzyme (PinELO) with very high homology to PirELO did not show activity with 20-carbon fatty acids. A series of chimeric proteins for these two enzymes were constructed to investigate the influence of different regions on substrate and product length. The substitution of a region from near the center of PirELO into PinELO resulted in an enzyme having EPA-elongating activity similar to that of PirELO. Only eight amino acids differed between the two proteins in this region; however, substitution of the same region from PinELO into PirELO produced a protein which was almost inactive. The addition of a small region from near the N-terminus of PinELO was sufficient to restore activity with GLA, indicating that amino acids from these two regions interact to determine protein structure or function. Predicted topology models for PirELO and PinELO placed the two regions described here near the luminal-proximal ends of the first and fourth/fifth transmembrane helixes, at the opposite end of the condensing enzyme from four conserved regions thought to form a catalytic ring. Thus, protein characteristics determined by specific luminal-proximal regions of fatty acid condensing enzymes have a major influence on substrate specificity and final product length.
Vrinten
Patricia L
PL
Bioriginal Food and Science Corporation, 110 Gymnasium Place, Saskatoon, Saskatchewan, Canada S7N0W9. pvrinten@bioriginal.ca
Hoffman
Travis
T
Bauer
Jörg
J
Qiu
Xiao
X
D000123:Acetyltransferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020418:Algal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020816:Amino Acid Motifs; D000081031:Fatty Acid Elongases; D005231:Fatty Acids, Unsaturated / Q000737:chemistry* / Q000378:metabolism; D011775:Pythium / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D020418:Algal Proteins; D005231:Fatty Acids, Unsaturated; D000123:Acetyltransferases; D000081031:Fatty Acid Elongases