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Identification and characterization of a novel hydroxylamine oxidase, DnfA, that catalyzes the oxidation of hydroxylamine to N(2)
Nitrogen (N(2)) gas in the atmosphere is partially replenished by microbial denitrification of ammonia. Recent study has shown that Alcaligenes ammonioxydans oxidizes ammonia to dinitrogen via a process featuring the intermediate hydroxylamine, termed “Dirammox” (direct ammonia oxidation). However,...
Autores principales: | , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society for Biochemistry and Molecular Biology
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9478400/ https://www.ncbi.nlm.nih.gov/pubmed/35970391 http://dx.doi.org/10.1016/j.jbc.2022.102372 |
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author | Wu, Meng-Ru Miao, Li-Li Liu, Ying Qian, Xin-Xin Hou, Ting-Ting Ai, Guo-Min Yu, Lu Ma, Lan Gao, Xi-Yan Qin, Ya-Ling Zhu, Hai-Zhen Du, Lei Li, Sheng-Ying Tian, Chang-Lin Li, De-Feng Liu, Zhi-Pei Liu, Shuang-Jiang |
author_facet | Wu, Meng-Ru Miao, Li-Li Liu, Ying Qian, Xin-Xin Hou, Ting-Ting Ai, Guo-Min Yu, Lu Ma, Lan Gao, Xi-Yan Qin, Ya-Ling Zhu, Hai-Zhen Du, Lei Li, Sheng-Ying Tian, Chang-Lin Li, De-Feng Liu, Zhi-Pei Liu, Shuang-Jiang |
author_sort | Wu, Meng-Ru |
collection | PubMed |
description | Nitrogen (N(2)) gas in the atmosphere is partially replenished by microbial denitrification of ammonia. Recent study has shown that Alcaligenes ammonioxydans oxidizes ammonia to dinitrogen via a process featuring the intermediate hydroxylamine, termed “Dirammox” (direct ammonia oxidation). However, the unique biochemistry of this process remains unknown. Here, we report an enzyme involved in Dirammox that catalyzes the conversion of hydroxylamine to N(2). We tested previously annotated proteins involved in redox reactions, DnfA, DnfB, and DnfC, to determine their ability to catalyze the oxidation of ammonia or hydroxylamine. Our results showed that none of these proteins bound to ammonia or catalyzed its oxidation; however, we did find DnfA bound to hydroxylamine. Further experiments demonstrated that, in the presence of NADH and FAD, DnfA catalyzed the conversion of (15)N-labeled hydroxylamine to (15)N(2). This conversion did not happen under oxygen (O(2))-free conditions. Thus, we concluded that DnfA encodes a hydroxylamine oxidase. We demonstrate that DnfA is not homologous to any known hydroxylamine oxidoreductases and contains a diiron center, which was shown to be involved in catalysis via electron paramagnetic resonance experiments. Furthermore, enzyme kinetics of DnfA were assayed, revealing a K(m) of 92.9 ± 3.0 μM for hydroxylamine and a k(cat) of 0.028 ± 0.001 s(−1). Finally, we show that DnfA was localized in the cytoplasm and periplasm as well as in tubular membrane invaginations in HO-1 cells. To the best of our knowledge, we conclude that DnfA is the first enzyme discovered that catalyzes oxidation of hydroxylamine to N(2). |
format | Online Article Text |
id | pubmed-9478400 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | American Society for Biochemistry and Molecular Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-94784002022-09-22 Identification and characterization of a novel hydroxylamine oxidase, DnfA, that catalyzes the oxidation of hydroxylamine to N(2) Wu, Meng-Ru Miao, Li-Li Liu, Ying Qian, Xin-Xin Hou, Ting-Ting Ai, Guo-Min Yu, Lu Ma, Lan Gao, Xi-Yan Qin, Ya-Ling Zhu, Hai-Zhen Du, Lei Li, Sheng-Ying Tian, Chang-Lin Li, De-Feng Liu, Zhi-Pei Liu, Shuang-Jiang J Biol Chem Research Article Nitrogen (N(2)) gas in the atmosphere is partially replenished by microbial denitrification of ammonia. Recent study has shown that Alcaligenes ammonioxydans oxidizes ammonia to dinitrogen via a process featuring the intermediate hydroxylamine, termed “Dirammox” (direct ammonia oxidation). However, the unique biochemistry of this process remains unknown. Here, we report an enzyme involved in Dirammox that catalyzes the conversion of hydroxylamine to N(2). We tested previously annotated proteins involved in redox reactions, DnfA, DnfB, and DnfC, to determine their ability to catalyze the oxidation of ammonia or hydroxylamine. Our results showed that none of these proteins bound to ammonia or catalyzed its oxidation; however, we did find DnfA bound to hydroxylamine. Further experiments demonstrated that, in the presence of NADH and FAD, DnfA catalyzed the conversion of (15)N-labeled hydroxylamine to (15)N(2). This conversion did not happen under oxygen (O(2))-free conditions. Thus, we concluded that DnfA encodes a hydroxylamine oxidase. We demonstrate that DnfA is not homologous to any known hydroxylamine oxidoreductases and contains a diiron center, which was shown to be involved in catalysis via electron paramagnetic resonance experiments. Furthermore, enzyme kinetics of DnfA were assayed, revealing a K(m) of 92.9 ± 3.0 μM for hydroxylamine and a k(cat) of 0.028 ± 0.001 s(−1). Finally, we show that DnfA was localized in the cytoplasm and periplasm as well as in tubular membrane invaginations in HO-1 cells. To the best of our knowledge, we conclude that DnfA is the first enzyme discovered that catalyzes oxidation of hydroxylamine to N(2). American Society for Biochemistry and Molecular Biology 2022-08-13 /pmc/articles/PMC9478400/ /pubmed/35970391 http://dx.doi.org/10.1016/j.jbc.2022.102372 Text en © 2022 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 Wu, Meng-Ru Miao, Li-Li Liu, Ying Qian, Xin-Xin Hou, Ting-Ting Ai, Guo-Min Yu, Lu Ma, Lan Gao, Xi-Yan Qin, Ya-Ling Zhu, Hai-Zhen Du, Lei Li, Sheng-Ying Tian, Chang-Lin Li, De-Feng Liu, Zhi-Pei Liu, Shuang-Jiang Identification and characterization of a novel hydroxylamine oxidase, DnfA, that catalyzes the oxidation of hydroxylamine to N(2) |
title | Identification and characterization of a novel hydroxylamine oxidase, DnfA, that catalyzes the oxidation of hydroxylamine to N(2) |
title_full | Identification and characterization of a novel hydroxylamine oxidase, DnfA, that catalyzes the oxidation of hydroxylamine to N(2) |
title_fullStr | Identification and characterization of a novel hydroxylamine oxidase, DnfA, that catalyzes the oxidation of hydroxylamine to N(2) |
title_full_unstemmed | Identification and characterization of a novel hydroxylamine oxidase, DnfA, that catalyzes the oxidation of hydroxylamine to N(2) |
title_short | Identification and characterization of a novel hydroxylamine oxidase, DnfA, that catalyzes the oxidation of hydroxylamine to N(2) |
title_sort | identification and characterization of a novel hydroxylamine oxidase, dnfa, that catalyzes the oxidation of hydroxylamine to n(2) |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9478400/ https://www.ncbi.nlm.nih.gov/pubmed/35970391 http://dx.doi.org/10.1016/j.jbc.2022.102372 |
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