Mechanistic insights into the dual activities of the single active site of L-lysine oxidase/monooxygenase from Pseudomonas sp. AIU 813

L-Lysine oxidase/monooxygenase (L-LOX/MOG) from Pseudomonas sp. AIU 813 catalyzes the mixed bioconversion of L-amino acids, particularly L-lysine, yielding an amide and carbon dioxide by an oxidative decarboxylation (i.e. apparent monooxygenation), as well as oxidative deamination (hydrolysis of oxidized product), resulting in a-keto acid, hydrogen peroxide (H₂O₂), and ammonia. Here, using high-resolution MS and monitoring transient reaction kinetics with stopped-flow spectrophotometry, we identified the products from the reactions of L-lysine and L-ornithine, indicating that besides decarboxylating imino acids (i.e. 5-aminopentanamide from L-lysine), L-LOX/ MOG also decarboxylates keto acids (5-aminopentanoic acid from L-lysine and 4-aminobutanoic acid from L-ornithine). The reaction of reduced enzyme and oxygen generated an imino acid and H₂O₂, with no detectable C4a-hydroperoxyflavin. Single-turnover reactions in which L-LOX/MOG was first reduced by L-lysine to form imino acid before mixing with various compounds revealed that under anaerobic conditions, only hydrolysis products are present. Similar results were obtained upon H₂O₂ addition after enzyme denaturation. H₂O₂ addition to active L-LOX/MOG resulted in formation of more 5-aminopen-tanoic acid, but not 5-aminopentamide, suggesting that H₂O₂ generated from L-LOX/MOG in situ can result in decarboxylation of the imino acid, yielding an amide product, and extra H₂O₂ resulted in decarboxylation only of keto acids. Molecular dynamics simulations and detection of charge transfer species suggested that interactions between the substrate and its binding site on L-LOX/MOG are important for imino acid decarboxylation. Structural analysis indicated that the flavoenzyme oxidases catalyzing decarboxylation of an imino acid all share a common plug loop configuration that may facilitate this decarboxylation.