Design and Evaluation of Biomimetic Methylene‐H4MPT Mimics

Tetrahydromethanopterin (H4MPT) is a specialized coenzyme found in methanogenic archaea and methylotrophic bacteria, essential for one‐carbon C1 transfer and redox reactions in processes like methanogenesis. However, its structural complexity and limited availability hinder its use in studying archaeal metabolism and H4MPT‐dependent enzymes. Inspired by the success of NAD(P)H biomimetic compounds, we employed a similar structural simplification strategy for methylene‐H4MPT, designing four mimics (mimics 1–4) that retain the crucial hydride‐donating imidazolidine unit while modifying the pterin structure and hydrophilic tail. Structural and crystallographic analysis of methylene‐H4MPT‐bound enzymes, including MtdA and Fae, highlighted the importance of the pterin group in hydrogen bonding and active site interactions. Activity evaluation of the mimics in hydride transfer assays and relay reactions with MtdA and NADPH‐dependent ketoreductase CgKR1 showed that the pterin structure is critical for enzymatic activity, and improving water solubility via hydrophilic tail modification enhances performance. This biomimetic approach offers functional substitutes for methylene‐H4MPT and potentially valuable tools for studying archaeal metabolism and synthetic microbial systems for C1 compound utilization.