In silico investigation of pretreatment-derived inhibitors on multiple methanogenic enzymes in anaerobic digestion: Molecular-level insights

Molecular docking is relatively underutilized in environmental microbiology, particularly, in the discourse of anaerobic digestion (AD). Multiple studies have confirmed that pre-treatment-produced inhibitors contribute to reduction in methane production. However, till today, limited studies offering molecular level mechanistic evidence against several enzymes that catalyses the pathways of AD demonstrating how these inhibitors interrupt the activity of methanogenic enzymes. Hence, the present study employed molecular docking, a computational chemistry approach, to elucidate enzyme-inhibitor interactions at the molecular level. In this study, nine (9) inhibitors (furfural, 5-hydroxymethylfurfural, vanillin, syringaldehyde, pyridine, pyrrole, pyrazine, levulinic acid and formic acid) were sourced from PubChem database, while four critical enzymes involved in the methanogenic stage were retrieved from Protein Data Bank (PDB) methyl-coenzyme M reductase (MCR), carbon monoxide dehydrogenase (CODH), heterodisulfide reductase (Hdr) and coenzyme M methyltransferase (MtaA). The study found that, among the nine inhibitors investigated, four (5-hydroxymethylfurfural, syringaldehyde, furfural and vanillin) of them exhibited strong enzyme-ligand interactions within the binding pockets of the target enzymes. Overall, the observed interactions between the studied inhibitors and enzymes were dominated by 25 hydrogen bond interactions, with two π–π stacking interactions contributing to ligand stabilization. MD simulations revealed that Hdr complexes maintained superior ligand occupancy and structural stability over the full 100 ns trajectory, while MCR complexes showed more transient inhibitor occupancy with periodic dissociation events. These findings provide molecular-level evidence of how pre-treatment-derived inhibitors interfere with the activity of methanogenic enzymes, contributing to reduced biomethane production. Overall, this research enhances understanding of the critical balance between improving substrate biodegradability through pre-treatment and minimizing the generation of inhibitory compounds.