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Mutation of a distal gating residue modulates NADH binding in NADH:Quinone oxidoreductase from Pseudomonas aeruginosa PAO1
Enzymes require flexible regions to adopt multiple conformations during catalysis. The mobile regions of enzymes include gates that modulate the passage of molecules in and out of the enzyme's active site. The enzyme PA1024 from Pseudomonas aeruginosa PA01 is a recently discovered flavin-depend...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society for Biochemistry and Molecular Biology
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10033279/ https://www.ncbi.nlm.nih.gov/pubmed/36803963 http://dx.doi.org/10.1016/j.jbc.2023.103044 |
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author | Moni, Bilkis Mehrin Quaye, Joanna A. Gadda, Giovanni |
author_facet | Moni, Bilkis Mehrin Quaye, Joanna A. Gadda, Giovanni |
author_sort | Moni, Bilkis Mehrin |
collection | PubMed |
description | Enzymes require flexible regions to adopt multiple conformations during catalysis. The mobile regions of enzymes include gates that modulate the passage of molecules in and out of the enzyme's active site. The enzyme PA1024 from Pseudomonas aeruginosa PA01 is a recently discovered flavin-dependent NADH:quinone oxidoreductase (NQO, EC 1.6.5.9). Q80 in loop 3 (residues 75–86) of NQO is ∼15 Å away from the flavin and creates a gate that seals the active site through a hydrogen bond with Y261 upon NADH binding. In this study, we mutated Q80 to glycine, leucine, or glutamate to investigate the mechanistic significance of distal residue Q80 in NADH binding in the active site of NQO. The UV-visible absorption spectrum reveals that the mutation of Q80 minimally affects the protein microenvironment surrounding the flavin. The anaerobic reductive half-reaction of the NQO-mutants yields a ≥25-fold increase in the K(d) value for NADH compared to the WT enzyme. However, we determined that the k(red) value was similar in the Q80G, Q80L, and wildtype enzymes and only ∼25% smaller in the Q80E enzyme. Steady-state kinetics with NQO-mutants and NQO-WT at varying concentrations of NADH and 1,4-benzoquinone establish a ≤5-fold decrease in the k(cat)/K(NADH) value. Moreover, there is no significant difference in the k(cat)/K(BQ) (∼1 × 10(6) M(−1)s(−1)) and k(cat) (∼24 s(−1)) values in NQO-mutants and NQO-WT. These results are consistent with the distal residue Q80 being mechanistically essential for NADH binding to NQO with minimal effect on the quinone binding to the enzyme and hydride transfer from NADH to flavin. |
format | Online Article Text |
id | pubmed-10033279 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | American Society for Biochemistry and Molecular Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-100332792023-03-24 Mutation of a distal gating residue modulates NADH binding in NADH:Quinone oxidoreductase from Pseudomonas aeruginosa PAO1 Moni, Bilkis Mehrin Quaye, Joanna A. Gadda, Giovanni J Biol Chem Research Article Collection: Enzymology Enzymes require flexible regions to adopt multiple conformations during catalysis. The mobile regions of enzymes include gates that modulate the passage of molecules in and out of the enzyme's active site. The enzyme PA1024 from Pseudomonas aeruginosa PA01 is a recently discovered flavin-dependent NADH:quinone oxidoreductase (NQO, EC 1.6.5.9). Q80 in loop 3 (residues 75–86) of NQO is ∼15 Å away from the flavin and creates a gate that seals the active site through a hydrogen bond with Y261 upon NADH binding. In this study, we mutated Q80 to glycine, leucine, or glutamate to investigate the mechanistic significance of distal residue Q80 in NADH binding in the active site of NQO. The UV-visible absorption spectrum reveals that the mutation of Q80 minimally affects the protein microenvironment surrounding the flavin. The anaerobic reductive half-reaction of the NQO-mutants yields a ≥25-fold increase in the K(d) value for NADH compared to the WT enzyme. However, we determined that the k(red) value was similar in the Q80G, Q80L, and wildtype enzymes and only ∼25% smaller in the Q80E enzyme. Steady-state kinetics with NQO-mutants and NQO-WT at varying concentrations of NADH and 1,4-benzoquinone establish a ≤5-fold decrease in the k(cat)/K(NADH) value. Moreover, there is no significant difference in the k(cat)/K(BQ) (∼1 × 10(6) M(−1)s(−1)) and k(cat) (∼24 s(−1)) values in NQO-mutants and NQO-WT. These results are consistent with the distal residue Q80 being mechanistically essential for NADH binding to NQO with minimal effect on the quinone binding to the enzyme and hydride transfer from NADH to flavin. American Society for Biochemistry and Molecular Biology 2023-02-18 /pmc/articles/PMC10033279/ /pubmed/36803963 http://dx.doi.org/10.1016/j.jbc.2023.103044 Text en © 2023 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Research Article Collection: Enzymology Moni, Bilkis Mehrin Quaye, Joanna A. Gadda, Giovanni Mutation of a distal gating residue modulates NADH binding in NADH:Quinone oxidoreductase from Pseudomonas aeruginosa PAO1 |
title | Mutation of a distal gating residue modulates NADH binding in NADH:Quinone oxidoreductase from Pseudomonas aeruginosa PAO1 |
title_full | Mutation of a distal gating residue modulates NADH binding in NADH:Quinone oxidoreductase from Pseudomonas aeruginosa PAO1 |
title_fullStr | Mutation of a distal gating residue modulates NADH binding in NADH:Quinone oxidoreductase from Pseudomonas aeruginosa PAO1 |
title_full_unstemmed | Mutation of a distal gating residue modulates NADH binding in NADH:Quinone oxidoreductase from Pseudomonas aeruginosa PAO1 |
title_short | Mutation of a distal gating residue modulates NADH binding in NADH:Quinone oxidoreductase from Pseudomonas aeruginosa PAO1 |
title_sort | mutation of a distal gating residue modulates nadh binding in nadh:quinone oxidoreductase from pseudomonas aeruginosa pao1 |
topic | Research Article Collection: Enzymology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10033279/ https://www.ncbi.nlm.nih.gov/pubmed/36803963 http://dx.doi.org/10.1016/j.jbc.2023.103044 |
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