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

• Published in: Protein science 2024-09, Vol.33 (9), p.e5144-n/a
• Main Authors: You, Cai, Pan, Yunjun, Liu, Ruxin, Li, Shengying, Feng, Yingang
• Format: Article
• Language: English
• Subjects: Autoimmune diseases; Biosynthesis; Catalytic Domain; crystal structure; Crystallography, X-Ray; Flexibility; Fungal Proteins - chemistry; Fungal Proteins - genetics; Fungal Proteins - metabolism; Homocysteine; Hydroxyl groups; Immunosuppressive agents; Methionine; Methylation; Methyltransferase; Methyltransferases - chemistry; Methyltransferases - genetics; Methyltransferases - metabolism; Models, Molecular; Mutagenesis; Mycophenolic acid; Mycophenolic Acid - chemistry; Mycophenolic Acid - metabolism; Natural products; O‐methyltransferase; Protein biosynthesis; Protein engineering; substrate flexibility; Substrate Specificity; Substrates
• ISSN: 0961-8368; 1469-896X; 1469-896X
• DOI: 10.1002/pro.5144

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.