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

© 2020 Trisrivirat et al. 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 deam...

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Main Authors: Duangthip Trisrivirat, Narin Lawan, Pirom Chenprakhon, Daisuke Matsui, Yasuhisa Asano, Pimchai Chaiyen
Format: Journal
Published: 2020
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spelling th-cmuir.6653943832-701902020-10-14T08:25:24Z Mechanistic insights into the dual activities of the single active site of l-lysine oxidase/monooxygenase from Pseudomonas sp. AIU 813 Duangthip Trisrivirat Narin Lawan Pirom Chenprakhon Daisuke Matsui Yasuhisa Asano Pimchai Chaiyen Biochemistry, Genetics and Molecular Biology © 2020 Trisrivirat et al. 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 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-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. 2020-10-14T08:25:24Z 2020-10-14T08:25:24Z 2020-08-07 Journal 1083351X 2-s2.0-85089300635 10.1074/jbc.RA120.014055 https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85089300635&origin=inward http://cmuir.cmu.ac.th/jspui/handle/6653943832/70190
institution Chiang Mai University
building Chiang Mai University Library
continent Asia
country Thailand
Thailand
content_provider Chiang Mai University Library
collection CMU Intellectual Repository
topic Biochemistry, Genetics and Molecular Biology
spellingShingle Biochemistry, Genetics and Molecular Biology
Duangthip Trisrivirat
Narin Lawan
Pirom Chenprakhon
Daisuke Matsui
Yasuhisa Asano
Pimchai Chaiyen
Mechanistic insights into the dual activities of the single active site of l-lysine oxidase/monooxygenase from Pseudomonas sp. AIU 813
description © 2020 Trisrivirat et al. 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 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-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.
format Journal
author Duangthip Trisrivirat
Narin Lawan
Pirom Chenprakhon
Daisuke Matsui
Yasuhisa Asano
Pimchai Chaiyen
author_facet Duangthip Trisrivirat
Narin Lawan
Pirom Chenprakhon
Daisuke Matsui
Yasuhisa Asano
Pimchai Chaiyen
author_sort Duangthip Trisrivirat
title Mechanistic insights into the dual activities of the single active site of l-lysine oxidase/monooxygenase from Pseudomonas sp. AIU 813
title_short Mechanistic insights into the dual activities of the single active site of l-lysine oxidase/monooxygenase from Pseudomonas sp. AIU 813
title_full Mechanistic insights into the dual activities of the single active site of l-lysine oxidase/monooxygenase from Pseudomonas sp. AIU 813
title_fullStr Mechanistic insights into the dual activities of the single active site of l-lysine oxidase/monooxygenase from Pseudomonas sp. AIU 813
title_full_unstemmed Mechanistic insights into the dual activities of the single active site of l-lysine oxidase/monooxygenase from Pseudomonas sp. AIU 813
title_sort mechanistic insights into the dual activities of the single active site of l-lysine oxidase/monooxygenase from pseudomonas sp. aiu 813
publishDate 2020
url https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85089300635&origin=inward
http://cmuir.cmu.ac.th/jspui/handle/6653943832/70190
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