Molecular mechanism of substrate specificity for delta 6 desaturase from Mortierella alpina and Micromonas pusilla.
The ω6 and ω3 pathways are two major pathways in the biosynthesis of PUFAs. In both of these, delta 6 desaturase (FADS6) is a key bifunctional enzyme desaturating linoleic acid or α-linolenic acid. Microbial species have different propensity for accumulating ω6- or ω3-series PUFAs, which may be determined by the substrate preference of FADS6 enzyme. In the present study, we analyzed the molecular mechanism of FADS6 substrate specificity. FADS6 cDNAs were cloned from Mortierella alpina (ATCC 32222) and Micromonas pusilla (CCMP1545) that synthesized high levels of arachidonic acid and EPA, respectively. M. alpina FADS6 (MaFADS6-I) showed substrate preference for LA; whereas, M. pusilla FADS6 (MpFADS6) preferred ALA. To understand the structural basis of substrate specificity, MaFADS6-I and MpFADS6 sequences were divided into five sections and a domain swapping approach was used to examine the role of each section in substrate preference. Our results showed that sequences between the histidine boxes I and II played a pivotal role in substrate preference. Based on our domain swapping results, nine amino acid (aa) residues were targeted for further analysis by site-directed mutagenesis. G194L, E222S, M227K, and V399I/I400E substitutions interfered with substrate recognition, which suggests that the corresponding aa residues play an important role in this process.
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, People's Republic of China.
Haisu
H
Shi
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, People's Republic of China Synergistic Innovation Center for Food Safety and Nutrition, Wuxi 214122, People's Republic of China haiqinchen@jiangnan.edu.cn yqchen@jiangnan.edu.cn.
Haiqin
H
Chen
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, People's Republic of China Synergistic Innovation Center for Food Safety and Nutrition, Wuxi 214122, People's Republic of China.
Zhennan
Z
Gu
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, People's Republic of China.
Yuanda
Y
Song
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, People's Republic of China Synergistic Innovation Center for Food Safety and Nutrition, Wuxi 214122, People's Republic of China.
Hao
H
Zhang
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, People's Republic of China Synergistic Innovation Center for Food Safety and Nutrition, Wuxi 214122, People's Republic of China.
Wei
W
Chen
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, People's Republic of China Synergistic Innovation Center for Food Safety and Nutrition, Wuxi 214122, People's Republic of China Department of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, NC 27157 haiqinchen@jiangnan.edu.cn yqchen@jiangnan.edu.cn.
Yong Q
YQ
Chen
D016718:Arachidonic Acid / Q000378:metabolism; D015118:Eicosapentaenoic Acid / Q000378:metabolism; D050602:Linoleoyl-CoA Desaturase / Q000378:metabolism*; D020094:Mortierella / Q000201:enzymology*; D013379:Substrate Specificity; D017962:alpha-Linolenic Acid / Q000378:metabolism
D017962:alpha-Linolenic Acid; D016718:Arachidonic Acid; D015118:Eicosapentaenoic Acid; D050602:Linoleoyl-CoA Desaturase
arachidonic acid; chimera; eicosapentaenoic acid; linoleic acid; polyunsaturated fatty acids; α-linolenic acid