Design, Synthesis and Activity Evaluation of 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 nicotinamide adenine dinucleotide (phosphate) (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 H₄MPT-dependent formaldehyde-activating enzyme MtdA and H₄MPT-dependent formaldehyde-activating enzyme 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.