Mutational and crystallographic analysis of <scp>l</scp>‐amino acid oxidase/monooxygenase from <i>Pseudomonas</i> sp. AIU 813: Interconversion between oxidase and monooxygenase activities

Matsui, Daisuke; Im, Dohyun; Sugawara, Asami; Fukuta, Yasuhisa; Fushinobu, Shinya; Isobe, Kimiyasu; Asano, Yasuhisa

doi:10.1016/j.fob.2014.02.002

Cited by 20 publications

(29 citation statements)

References 55 publications

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“…In the ligand‐free structure, a region connecting β16 and α15 (Met419–His428) was not included in the final model due to disorder (Fig. A, red and blue dotted lines) . In the three complex structures determined in this study, this region was clearly observed as a 2‐turn α‐helix (Fig.…”

Section: Discussionmentioning

confidence: 96%

“…AIU 813 ( l ‐AAO/MOG) is a bifunctional enzyme that exhibits both the l ‐Lys α‐oxidase and l ‐Lys 2‐monooxygenase (EC http://www.chem.qmul.ac.uk/iubmb/enzyme/EC1/13/12/2.html) activities (Fig. ) . Most l ‐AAOs show a wide range of substrate specificity, as represented by snake venom l ‐AAOs .…”

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confidence: 99%

“…The l ‐AAOs are FAD‐dependent enzymes and members of the flavin‐containing monoamine oxidase (MAO) family . l ‐AAO/MOG is classified into a distinct phylogenetic branch from other MAO family enzymes along with PAO and TMO . However, l ‐AAO/MOG shows low or barely detectable amino acid sequence identity to these enzymes: 16.7% to LGOX, 18.8% to PAO, and 30.1% to TMO by EMBOSS Needle pairwise alignment.…”

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confidence: 99%

“…Relative oxidase activities of l ‐AAO/MOG toward l ‐ornithine (Orn) and l ‐Arg compared with l ‐Lys were 31% and 6%, respectively . In our previous study , a ligand‐free crystal structure of l ‐AAO/MOG and its interconversion between oxidase and monooxygenase activities by chemical modification and point mutation were described. The key residue for the activity conversion (Cys254) is located behind the aromatic substrate‐binding cage but not directly involved in the interaction with the substrate.…”

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confidence: 99%

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Ligand complex structures of l‐amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813 and its conformational change

Matsui

Arakawa

et al. 2018

FEBS Open Bio

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l ‐Amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813 ( l ‐ AAO / MOG ) catalyzes both the oxidative deamination and oxidative decarboxylation of the α‐group of l ‐Lys to produce a keto acid and amide, respectively. l ‐ AAO / MOG exhibits limited specificity for l ‐amino acid substrates with a basic side chain. We previously determined its ligand‐free crystal structure and identified a key residue for maintaining the dual activities. Here, we determined the structures of l ‐ AAO / MOG complexed with l ‐Lys, l ‐ornithine, and l ‐Arg and revealed its substrate recognition. Asp238 is located at the ceiling of a long hydrophobic pocket and forms a strong interaction with the terminal, positively charged group of the substrates. A mutational analysis on the D238A mutant indicated that the interaction is critical for substrate binding but not for catalytic control between the oxidase/monooxygenase activities. The catalytic activities of the D238E mutant unexpectedly increased, while the D238F mutant exhibited altered substrate specificity to long hydrophobic substrates. In the ligand‐free structure, there are two channels connecting the active site and solvent, and a short region located at the dimer interface is disordered. In the l ‐Lys complex structure, a loop region is displaced to plug the channels. Moreover, the disordered region in the ligand‐free structure forms a short helix in the substrate complex structures and creates the second binding site for the substrate. It is assumed that the amino acid substrate enters the active site of l ‐ AAO / MOG through this route. Database The atomic coordinates and structure factors (codes 5YB6 , 5YB7 , and 5YB8 ) have been deposited in the Protein Data Bank ( http://wwpdb.org/ ). EC numbers 1.4.3.2 ( l ‐amino acid oxidase), 1.13.12.2 (lysine 2‐monooxygenase).

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Section: Discussionmentioning

confidence: 96%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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Ligand complex structures of l‐amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813 and its conformational change

Matsui

Arakawa

et al. 2018

FEBS Open Bio

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“…When the mutant plasmids of pUC19zeoR-lodA were introduced into E. coli BL21 (DE3) harboring pCDF-lodB, we obtained about 100 phleomycin-resistant colonies from approximately 2 × 10 5 ampicillin-resistant colonies (the ratio was about 0.2%), and these phleomycin-resistant colonies were screened for LodA activity using a color development method with 4-aminoantipyrine, Nethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline, and peroxidase. 16) One mutant showing signs of LodA activity was obtained by this screening method, and it is suggested that ZeoR can be used as a fusion reporter for the selection. The selected strain produced the LodA in the cells by cultivation at 25°C for 24 h in the LBAPSI medium, and its enzyme activity was 0.17 U/mL of the culture without the removal of E. coli catalase by ethanol precipitation (the specific activity of the crude extract was 0.63 U/mg of protein).…”

Section: Screening Of Active Loda Variant By Fusion With Zeormentioning

confidence: 99%

Heterologous production of l-lysine ε-oxidase by directed evolution using a fusion reporter method

Matsui

Asano

2015

Bioscience, Biotechnology, and Biochemistry

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For the heterologous production of l-lysine ε-oxidase (LodA), we constructed a new plasmid carrying LodA gene fused in-frame with an antibiotic (phleomycine) resistant gene. The new plasmid was randomly mutated and the mutated plasmids were transformed into Escherichia coli BL21 (DE3) harboring lodB, which encodes a protein (LodB) acting in posttranslational modification of LodA, and active mutants were selected by phleomycin resistance and oxidase activities. One soluble LodA variant isolated by this method contained six silent mutations and one missense mutation. At these mutation points, the codon adaptations at Lys92, Ala550, and Thr646, and the amino acid substitution at His286 to Arg contributed to the production of its functional form. The active form of LodA variant was induced by post-modification of LodB in the heterologous coexpression, and the activity increased with additional NaCl and heat treatment. This is the first report of heterologous production of LodA by random mutagenesis.

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Flavoprotein Monooxygenases and Halogenases

Paul

Guarneri

Berkel

2021

Flavin‐Based Catalysis

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Article 25fa states that the author of a short scientific work funded either wholly or partially by Dutch public funds is entitled to make that work publicly available for no consideration following a reasonable period of time after the work was first published, provided that clear reference is made to the source of the first publication of the work.This publication is distributed under The Association of Universities in the Netherlands (VSNU) 'Article 25fa implementation' project. In this project research outputs of researchers employed by Dutch Universities that comply with the legal requirements of Article 25fa of the Dutch Copyright Act are distributed online and free of cost or other barriers in institutional repositories. Research outputs are distributed six months after their first online publication in the original published version and with proper attribution to the source of the original publication.

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Mutational and crystallographic analysis of l‐amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813: Interconversion between oxidase and monooxygenase activities

Cited by 20 publications

References 55 publications

Ligand complex structures of l‐amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813 and its conformational change

Ligand complex structures of l‐amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813 and its conformational change

Heterologous production of l-lysine ε-oxidase by directed evolution using a fusion reporter method

Flavoprotein Monooxygenases and Halogenases

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