Functional and structural characterization of the human indolethylamine N-methyltransferase through fluorometric, thermal and computational docking analyses.

Indolethylamine N-methyltransferase (INMT) is a key enzyme in the biosynthesis of compounds like the psychedelic DMT, yet its structure and mechanism remain poorly understood. This work presents the first fluorometric steady-state assay for human INMT using quinoline as a substrate, validated by thermal shift and docking analyses. The enzyme is unstable, requiring acidic or near-neutral pH and low salt. The assay yields kinetic constants comparable to other methyltransferases, and docking shows quinoline binds at the same site as tryptamine. This method offers a simpler alternative to radioactive or mass spectrometry assays, enabling reliable kinetic studies and future investigations into enzyme mutants linked to psychiatric disorders and cancer.

Study at a glance

Abstract

The "psychedelic renaissance" is sparking growing interest in clinical research, along with a rise in clinical trials. Substances such as 3,4-methylenedioxymethamphetamine (MDMA), psilocybin and N,N-dimethyltryptamine (DMT) are involved. The focus of this paper is on indolethylamine N-methyltransferase (INMT), a crucial enzyme in the biosynthesis of key compounds, including DMT, which meets science, medicine and spirituality. The presence of DMT in animals and plants raises many questions about its biological role. Meanwhile, the distribution of INMT in various organs and its involvement in diseases like cancer and mental disorders also fuel investigations worldwide. However, INMT remains largely unexplored, particularly its enzymatic mechanism and structural properties, leaving a significant gap in potential applications. This study examines for the first time the catalytic activity of the human INMT (hINMT) using a simple fluorometric steady-state assay employing the substrate quinoline. The findings are supported by thermal shift and docking analyses, providing valuable information about optimal chemical conditions and potential binding sites for substrates. The thermal shift assays indicate that recombinant hINMT is unstable and requires acidic or near-neutral pH and low salt levels. These experiments also allow for the estimation of dissociation constants for its natural coenzymes SAM and SAH, helping to determine the appropriate setup for the fluorometric assays and calculate kinetic constants, which are comparable to other methyltransferases. The docking indicates that quinoline occupies the same site as the natural substrate tryptamine, further validating the fluorometric approach. The paper provides a foundation for thoroughly studying hINMT under consistent conditions, which is crucial for obtaining reliable kinetic data and maintaining molecular stability for future structural analysis. This represents a valid alternative over previous endpoint radioactive-based and chromatography-mass spectrometry assays, which can provide only apparent steady-state parameters. Given the polymorphisms observed in hINMT and their potential association with psychiatric disorders, e.g., schizophrenia, and cancer, this strategy could serve as an invaluable tool for understanding the structure-function relationship of enzyme mutants and their role in diseases. Furthermore, these findings for the first time provide insights into the interaction modalities of hINMT with its substrates and lay the groundwork for inhibition experiments aimed at practical applications.