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.
Zhang
Xinxin
X
Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Department of Microbiology, Nankai University, Tianjin, PR China.
Wei
Dongsheng
D
Li
Mingchun
M
Qi
Yuyan
Y
Xing
Laijun
L
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002849:Chromatography, Gas; D019143:Evolution, Molecular; D044943:Fatty Acid Desaturases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D000596:Amino Acids; D011994:Recombinant Proteins; D044943:Fatty Acid Desaturases