Evolution-related amino acids play important role in determining regioselectivity of fatty acid desaturase from Pichia pastoris.

Omega(3)-fatty acid desaturase and Delta(12)-fatty acid desaturase of Pichia pastoris with distinguishable regioselectivity and high degree of sequence similarity were chosen for regioselectivity research. Chimeras were constructed in which Histidine-rich boxes 1, 2 and the carboxyl terminal region of omega(3)-fatty acid desaturase were replaced with corresponding region of Delta(12)-fatty acid desaturase. The replacement was found to result in a change of regioselectivity from omegay to x + 3 by functionally characterizing these chimeric enzymes in Saccharomyces cerevisae strain INVScI. Using site-directed mutagenesis, we further demonstrated that seven conserved amino acids of omega(3)-fatty acid desaturase within the first two Histidine-rich regions are responsible for the regioselectivity switch. Therefore, the regioselectivity of fatty acid desaturases may be better understood by investigating the evolutionary relationships of different fatty acid desaturases.