Search

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. [ABSTRACT FROM AUTHOR]

Histone methylation is associated with transcription regulation, but its role for glycoside hydrolase (GH) biosynthesis is still poorly understood. We identified the histone H3 lysine 79 (H3K79)-specific methyltransferase Po Dot1 in Penicillium oxalicum. Po Dot1 affects conidiation by regulating the transcription of key regulators (BrlA, FlbC, and StuA) of asexual development and is required in normal hyphae septum and branch formation by regulating the transcription of five septin-encoding genes, namely, aspA , aspB , aspC , aspD , and aspE. Tandem affinity purification/mass spectrometry showed that Po Dot1 has no direct interaction with transcription machinery, but it affects the expressions of extracellular GH genes extensively. The expression of genes (amy15A , amy13A , cel7A/cbh1 , cel61A , chi18A , cel3A/bgl1 , xyn10A , cel7B/eg1 , cel5B/eg2 , and cel6A/cbh2) that encode the top 10 GHs was remarkably downregulated by Podot1 deletion (Δ Podot1). Consistent with the decrease in gene transcription level, the activities of amylases and cellulases were significantly decreased in Δ Podot1 mutants in agar (solid) and fermentation (liquid) media. The repression of GH gene expressions caused by Po Dot1 deletion was not mediated by key transcription factors, such as AmyR, ClrB, CreA, and XlnR, but was accompanied by defects in global demethylated H3K79 (H3K79me2) and trimethylated H3K79 (H3K79me3). The impairment of H3K79me2 on specific GH gene loci was observed due to Po Dot1 deletion. The results implies that defects of H3K79 methylation is the key reason of the downregulated transcription level of GH-encoding genes and reveals the indispensable role of PoDot1 in extracellular GH biosynthesis. [ABSTRACT FROM AUTHOR]

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., (© 2024 The Protein Society.)