Enzymatic C3-Methylation of Indoles Using Methyltransferase PsmD─Crystal Structure, Catalytic Mechanism, and Preparative Applications

Abstract

Enantioselective methylation is a challenging task in organic chemistry, yet often desirable in drug discovery and optimization. S-Adenosyl methionine (SAM)-dependent methyltransferases (MTases) offer a selective alternative to chemical synthesis and an abundance of potential scaffolds. The crystal structure of C3-indole MTase PsmD from Streptomyces griseofuscus, involved in the biosynthesis of the acetylcholinesterase inhibitor physostigmine, was determined via X-ray crystallography. The amino acid residues essential for catalysis were identified by site-directed mutagenesis, and a mechanism of action was proposed. Furthermore, a PsmD ortholog was identified and characterized. The variant catalyzed enantioselective C-methylation over a broad substrate scope while displaying increased stability. Using this enzyme, preparative-scale enzymatic methylation was performed in cell-free extracts in combination with an SAM recycling system, eliminating the need for cofactor supplementation.

Application of the C3-Methyltransferase StspM1 for the Synthesis of the Natural Pyrroloindole Motif

“…The substrate binding modes were predicted by flexible docking with Glide. , To determine the oligomerization state of StspM1, size exclusion chromatography was performed. The deviation of the measured weight (70.5 kDa; Table S5 and Figure S8) from the theoretical size of a dimer (61.8 kDa) could be explained by the nonperfectly globular shape of the dimer and has also been observed for the homologous PsmD methyltransferase, which is proven to be a dimer via the crystal structure . Furthermore, a dimeric structure was also predicted by GalaxyHomomer .…”

“…To determine the substrate conversion rate, the commercially available bioluminescence-based Mtase-Glo Assay (Promega) was used, as previously reported. 24 This assay, which detects the formation of SAH, was used to measure the consumption of SAM in the methyltransferase reaction. 25 Plotting the reaction rate against the concentrations, K m values of 3.86 μM for LL-cWW 7a and 14.75 μM for its enantiomer DD-cWW 7c were measured, indicating a higher affinity of the enzyme for LL-cWW.…”

“…The methylation reaction was stopped after 5, 10, and 15 min by addition of 0.5% trifluoroacetic acid. To determine the substrate conversion rate, the commercially available bioluminescence-based Mtase-Glo Assay (Promega) was used, as previously reported . This assay, which detects the formation of SAH, was used to measure the consumption of SAM in the methyltransferase reaction …”

“…The deviation of the measured weight (70.5 kDa; Table S5 and Figure S8) from the theoretical size of a dimer (61.8 kDa) could be explained by the nonperfectly globular shape of the dimer and has also been observed for the homologous PsmD methyltransferase, which is proven to be a dimer via the crystal structure. 24 Furthermore, a dimeric structure was also predicted by GalaxyHomomer. 30 The presence of solvent-exposed apolar amino acids in the β-cap domain suggests a plausible dimerization mediated by this domain.…”

Advanced exploitation of unmerged reflection data during processing and refinement with autoPROC and BUSTER

“…Radiation damage at the ligand. The deposited data for PDB entry 7zkg (Amariei et al, 2022) contain the scaled and unmerged reflection data that allow the generation of a set of F(early) and F(late) amplitudes (using the first 195 and last 219 images, respectively) in addition to the amplitudes after merging reflections from all 3600 images. After BUSTER refinement, the F(early) À F(late) difference map shows some very pronounced positive peaks (loss of electrons) for several Asp and Glu carboxyl groups.…”