Structural basis for substrate flexibility of the O-methyltransferase MpaG' involved in mycophenolic acid biosynthesis

被引:0
|
作者
You, Cai [1 ,2 ,3 ]
Pan, Yunjun [2 ]
Liu, Ruxin [2 ]
Li, Shengying [2 ,4 ]
Feng, Yingang [1 ,3 ,5 ,6 ,7 ]
机构
[1] Chinese Acad Sci, Qingdao Inst Bioenergy & Bioproc Technol, CAS Key Lab Biofuels, Shandong Prov Key Lab Synthet Biol, Qingdao 266101, Shandong, Peoples R China
[2] Shandong Univ, State Key Lab Microbial Technol, Qingdao 266237, Shandong, Peoples R China
[3] Shandong Energy Inst, Qingdao, Shandong, Peoples R China
[4] Qingdao Natl Lab Marine Sci & Technol, Lab Marine Biol & Biotechnol, Qingdao, Shandong, Peoples R China
[5] Qingdao New Energy Shandong Lab, Qingdao, Shandong, Peoples R China
[6] Chinese Acad Sci, Qingdao Inst Bioenergy & Bioproc Technol, Shandong Engn Lab Single Cell Oil, Qingdao, Shandong, Peoples R China
[7] Univ Chinese Acad Sci, Beijing, Peoples R China
基金
中国博士后科学基金; 中国国家自然科学基金;
关键词
crystal structure; mycophenolic acid; O-methyltransferase; protein engineering; substrate flexibility; SPECIFICITY; GENE;
D O I
10.1002/pro.5144
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
MpaG' is an S-adenosyl-L-methionine (SAM)-dependent methyltransferase involved in the compartmentalized biosynthesis of mycophenolic acid (MPA), a first-line immunosuppressive drug for organ transplantations and autoimmune diseases. MpaG' catalyzes the 5-O-methylation of three precursors in MPA biosynthesis including demethylmycophenolic acid (DMMPA), 4-farnesyl-3,5-dihydroxy-6-methylphthalide (FDHMP), and an intermediate containing three fewer carbon atoms compared to FDHMP (FDHMP-3C) with different catalytic efficiencies. Here, we report the crystal structures of S-adenosyl-L-homocysteine (SAH)/DMMPA-bound MpaG', SAH/FDHMP-3C-bound MpaG', and SAH/FDHMP-bound MpaG' to understand the catalytic mechanism of MpaG' and structural basis for its substrate flexibility. Structural and biochemical analyses reveal that MpaG' utilizes the catalytic dyad H306-E362 to deprotonate the C5 hydroxyl group of the substrates for the following methylation. The three substrates with differently modified farnesyl moieties are well accommodated in a large semi-open substrate binding pocket with the orientation of their phthalide moiety almost identical. Based on the structure-directed mutagenesis, a single mutant MpaG'Q267A is engineered with significantly improved catalytic efficiency for all three substrates. This study expands the mechanistic understanding and the pocket engineering strategy for O-methyltransferases involved in fungal natural product biosynthesis. Our research also highlights the potential of O-methyltransferases to modify diverse substrates by protein design and engineering.
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页数:14
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