Structure-function analysis of access engineering in an FMNH₂-dependent monooxygenase for enhanced polyethylene...

Enzymatic functionalization of polyethylene is fundamentally limited by a backbone consisting entirely of inert CH bonds, and a structural framework for enzyme-mediated PE oxidation remains limited. Here we identify PA2355 (FMO), a soluble FMNH₂-dependent monooxygenase from Pseudomonas aeruginosa that hydroxylates untreated polyethylene in a purified, cell-free system. Eight crystal structures of the wild type enzyme and engineered variants in complex with ligands of increasing size reveal a compact active site in which a single residue Y393, simultaneously anchors the FMN cofactor and blocks substrate access. The wild type pocket accommodates molecules up to pentane (C5) but excludes diethylene glycol, which binds only outside the cavity. Replacing Y393 with alanine opens a continuous hydrophobic tunnel that the same molecule excluded in the wild type now enters and occupies the catalytic cavity in Y393A, and DiffDock based docking threads eicosane (C20) along the reactive flavin edge. Y393A accelerates PE surface hydroxylation from detectable at 21 days (wild type) to 3 days, validated by FT-IR, XPS, contact angle analysis and GC-MS. Molecular dynamics simulations support recurring configurations compatible with catalysis. These results support "access engineering", the targeted removal of steric barriers to unlock latent enzymatic activities on macromolecular substrates.