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| Hauptseite > Publikationsdatenbank > Indole C-methyltransferases – creating an efficient platform for the enantioselective methylation of bioactive compounds |
| Hauptseite > Publikationsdatenbank > Indole C-methyltransferases – creating an efficient platform for the enantioselective methylation of bioactive compounds |
| Book/Dissertation / PhD Thesis | FZJ-2026-01795 |
2026
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
Jülich
ISBN: 978-3-95806-881-0
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Please use a persistent id in citations: urn:nbn:de:0001-2606291408367.917935050069 doi:10.34734/FZJ-2026-01795
Abstract: The biosynthesis of the acetylcholinesterase (AChE) inhibitor physostigmine was described for the producing organism Streptomyces griseofuscus, and it was found to include a stereoselective methylation step catalyzed by the SAM-dependent methyltransferase PsmD, which determines the configuration of the final compound and triggers the formation of the specific pyrroloindole ring. Several PsmD product analogs have already demonstrated AChE inhibition, making the structural diversification of PsmD products an intriguing prospect, as it provides potential for identifying new drug candidates to treat related neurological disorders. A new PsmD-like indole C-methyltransferase was identified, originating from Streptomyces albulus (noursei) (PsmD_Sa). The enzyme was expressed and analyzed regarding structure, mechanism and biochemical properties. The biochemical analysis revealed a higher stability, compared to its previously characterized homolog from Streptomyces griseofuscus (PsmD_Sg), while maintaining the stereoselectivity of the methylation reaction. The crystal structure of PsmD_Sg was determined experimentally using X-ray spectroscopy in collaboration with Prof. Dr. Oliver Weiergräber (IBI-7: Structural biochemistry, Forschungszentrum Jülich) and used as a template for the precise in silico modeling of the PsmD_Sa structure. Site-directed mutagenesis was used to map the catalytic site and elucidate the mechanism of PsmD_Sa. The results were corroborated with molecular docking and molecular dynamic simulations performed by Dr. Benoit David (IBG-4: Bioinformatics, Forschungszentrum Jülich), offering an overview of the PsmD catalysis. A mobile N-terminal the substrate and the cofactor. A Glu-His-Tyr catalytic triad was found to activate the substrate through a proton shuttling action. The semi-rational engineering of PsmD_Sa led to the improvement of its activity towards bulky non-natural indole-containing substrates. Five positions in the catalytic site were targeted in a sequential manner through site saturation mutagenesis, producing focused mutant libraries. To generate and screen the resulting mutant libraries, the AutoBioTech integrated laboratory platform was utilized in collaboration with Dr. Julia Tenhaef (IBG-1: Biotechnology, Forschungszentrum Jülich). A modular approach was designed to automate the enzyme expression, enzymatic reactions and activity screening in 96-well microtiter plates. A new highthroughput colorimetric assay for indole detection was developed for the efficient screening of the resulting mutant libraries. The assay allows the detection of the PsmD substrate concentration in the presence of the isolated enzyme, as well as using whole-cell biocatalysts. The engineering and screening of the PsmD_Sa variants led to the identification of new mutants with significantly improved activity for the tested substrates, and the key position 166 was found to play an important role in the productive binding of bulky substrate derivatives. The site-directed mutagenesis of PsmD_Sa aimed to expand its alkylation capacity using an ethylated SAM cofactor derivative (SAE). Two enzymatic SAE supply systems were used in cascade with PsmD_Sa and the new mutants. The coupled PsmD_SAE supply systems were optimized, leading to a 7-fold improvement of PsmD-catalyzed conversion to the ethylated product using the wild-type enzyme. Two mutants, A125G and F126L performed better than the wild type in the reactions using several SAM cofactor derivatives. Finally, the preparative enzymatic methylation catalyzed by PsmD_Sa was achieved using different enzyme formulations: lysates, whole cells and immobilized enzymes. The reactions were carried out in combination with a cofactor recycling system and the stereoselective methylation was successfully achieved using various substrates and enzyme variants, in scales up to hundreds of milligrams. Overall, this study aimed to provide new insights into the practical aspects of methyltransferase biocatalysis and showcase the potential of PsmD_Sa as a useful tool for the stereoselective C-methylation of indole derivatives.
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