Zhang Xinxin, Wei Dongsheng, Li Mingchun, Qi Yuyan, Xing Laijun
Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Department of Microbiology, Nankai University, Tianjin, PR China.
Mol Biol Rep. 2009 Mar;36(3):567-73. doi: 10.1007/s11033-008-9215-6. Epub 2008 Feb 20.
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.
选择毕赤酵母中具有可区分区域选择性和高度序列相似性的Omega(3)-脂肪酸去饱和酶和Delta(12)-脂肪酸去饱和酶进行区域选择性研究。构建嵌合体，其中Omega(3)-脂肪酸去饱和酶的富含组氨酸的框1、2和羧基末端区域被Delta(12)-脂肪酸去饱和酶的相应区域取代。通过在酿酒酵母菌株INVScI中对这些嵌合酶进行功能表征，发现这种取代导致区域选择性从omega3变为x + 3。使用定点诱变，我们进一步证明，在前两个富含组氨酸的区域内，Omega(3)-脂肪酸去饱和酶的七个保守氨基酸负责区域选择性转换。因此，通过研究不同脂肪酸去饱和酶的进化关系，可以更好地理解脂肪酸去饱和酶的区域选择性。
