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

Abstract

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 α-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 yielding 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-aminopentanoic 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.

Catalytic mechanism of ancestral L-lysine oxidase assigned by sequence data mining

“…3). As shown in a previous study (41), 5-APNA was produced under the conditions containing H2O2 by hydrolyzing and decarboxylating the resultant imino acids (Fig. 3).…”

“…3), suggesting that, for AncLLysO, the monooxygenase function was the promiscuous activity compared with the oxidase function. This was clearly different in the case of L-LOX/MOG, where the monooxygenase function was the main activity identified through LC-HRMS analysis (41).…”

“…The mechanism generally corresponds with that of LAAOs (4). On the other hand, 5-APNM is produced by the monooxygenase activity of AncLLysO via a mechanism similar to that of L-LOX/MOG (41). Utilizing H2O2, imino acid is decarboxylated to 5-APNM at the active site (d to e in Fig.…”

“…The assigned LLysO has low sequence identity (less than 30%) to the previously reported LLysO from Trichoderma viridae (TvLysOX) (37,38) and L-amino acid oxidase/monooxygenase (L-LOX/MOG) from Pseudomonas sp. AIU813 (39)(40)(41); therefore, the enzymatic properties of the assigned LLysO could not be estimated only from the sequence analysis. Ancestral LLysO (AncLLysO) was designed utilizing the assigned LLysO sequences as templates.…”

Comprehensive metabolomic profiling of osteosarcoma based on UHPLC-HRMS

“…Lysine was thought to be a nonspecific bridge molecule, that could associate antigen with T cells, causing T cells to produce a specific effect against the antigen. 5-aminopentanamide, as a product of the decarboxylation of lysine (Trisrivirat et al 2020 ), may be related to the aberrant immune response in tumor patients. Our findings provide potential biomarkers for the timely discovery of lung metastases.…”