Issue 21, 2024

Directed evolution of C-methyltransferase PsmD for enantioselective pyrroloindole derivative production

Abstract

The natural product physostigmine is known for its capacity to inhibit acetylcholinesterase (AChE). The pyrroloindole-based scaffold of physostigmine is prevalent among various compounds demonstrating AChE inhibition, suggesting that its structural diversification holds promise as a strategy for the development of novel AChE inhibitors. The C-methyltransferase PsmD is involved in the biosynthesis of physostigmine. While the two described variants from Streptomyces griseofuscus and Streptomyces albulus display an extended substrate range, their specificity hinders the efficient methylation of substrate derivatives. In order to improve the activity of PsmD towards voluminous non-natural substrates, we employed an iterative saturation mutagenesis strategy, which led to an increase in the available space in the catalytic site, while maintaining stereoselectivity. To aid our efforts and provide an efficient platform for the evolution of pyrroloindole-forming enzymes, we developed a modular automated process for the expression, enzymatic reaction and activity screening of the obtained mutant libraries, using an integrated robotic system. In this way, we identified multiple mutants, which led to increased specific activity towards our target substrates. Our results enabled the identification of amino acid position 166 as a key site for the modulation of substrate specificity. We immobilized the best mutant W166C, and used it for the preparative synthesis of an AChE inhibitor, in the presence of a SAM cofactor recycling system.

Graphical abstract: Directed evolution of C-methyltransferase PsmD for enantioselective pyrroloindole derivative production

Supplementary files

Article information

Article type
Paper
Submitted
23 May 2024
Accepted
05 Sep 2024
First published
05 Sep 2024
This article is Open Access
Creative Commons BY license

Catal. Sci. Technol., 2024,14, 6298-6306

Directed evolution of C-methyltransferase PsmD for enantioselective pyrroloindole derivative production

D. A. Amariei, J. Tenhaef, T. Classen, B. David, T. M. Rosch, H. Gohlke, S. Noack and J. Pietruszka, Catal. Sci. Technol., 2024, 14, 6298 DOI: 10.1039/D4CY00657G

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