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Short-Chain Dehydrogenase NcmD Is Responsible for the C-10 Oxidation of Nocamycin F in Nocamycin Biosynthesis
Nocamycins I and II, featured with a tetramic acid scaffold, were isolated from the broth of Saccharothrix syringae NRRL B-16468. The biosynthesis of nocamycin I require an intermediate bearing a hydroxyl group at the C-10 position. A short chain dehydrogenase/reductase NcmD was proposed to catalyze...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2020
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773637/ https://www.ncbi.nlm.nih.gov/pubmed/33391238 http://dx.doi.org/10.3389/fmicb.2020.610827 |
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author | Mo, Xuhua Zhang, Hui Du, Fengyu Yang, Song |
author_facet | Mo, Xuhua Zhang, Hui Du, Fengyu Yang, Song |
author_sort | Mo, Xuhua |
collection | PubMed |
description | Nocamycins I and II, featured with a tetramic acid scaffold, were isolated from the broth of Saccharothrix syringae NRRL B-16468. The biosynthesis of nocamycin I require an intermediate bearing a hydroxyl group at the C-10 position. A short chain dehydrogenase/reductase NcmD was proposed to catalyze the conversion of the hydroxyl group to ketone at the C-10 position. By using the λ-RED recombination technology, we generated the NcmD deletion mutant strain S. syringae MoS-1005, which produced a new intermediate nocamycin F with a hydroxyl group at C-10 position. We then overexpressed NcmD in Escherichia coli BL21 (DE3), purified the His(6)-tagged protein NcmD to homogeneity and conducted in vitro enzymatic assays. NcmD showed preference to the cofactor NAD(+), and it effectively catalyzed the conversion from nocamyin F to nocamycin G, harboring a ketone group at C-10 position. However, NcmD showed no catalytic activity toward nocamyin II. NcmD achieved maximum catalytic activity at 45°C and pH 8.5. The kinetics of NcmD toward nocamycin F was investigated at 45°C, pH 8.5 in the presence of 2 mM NAD(+). The K(m) and k(cat) values were 131 ± 13 μM and 65 ± 5 min(−1), respectively. In this study, we have characterized NcmD as a dehydrogenase, which is involved in forming the ketone group at the C-10 position of nocamycin F. The results provide new insights to the nocamycin biosynthetic pathway. |
format | Online Article Text |
id | pubmed-7773637 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-77736372021-01-01 Short-Chain Dehydrogenase NcmD Is Responsible for the C-10 Oxidation of Nocamycin F in Nocamycin Biosynthesis Mo, Xuhua Zhang, Hui Du, Fengyu Yang, Song Front Microbiol Microbiology Nocamycins I and II, featured with a tetramic acid scaffold, were isolated from the broth of Saccharothrix syringae NRRL B-16468. The biosynthesis of nocamycin I require an intermediate bearing a hydroxyl group at the C-10 position. A short chain dehydrogenase/reductase NcmD was proposed to catalyze the conversion of the hydroxyl group to ketone at the C-10 position. By using the λ-RED recombination technology, we generated the NcmD deletion mutant strain S. syringae MoS-1005, which produced a new intermediate nocamycin F with a hydroxyl group at C-10 position. We then overexpressed NcmD in Escherichia coli BL21 (DE3), purified the His(6)-tagged protein NcmD to homogeneity and conducted in vitro enzymatic assays. NcmD showed preference to the cofactor NAD(+), and it effectively catalyzed the conversion from nocamyin F to nocamycin G, harboring a ketone group at C-10 position. However, NcmD showed no catalytic activity toward nocamyin II. NcmD achieved maximum catalytic activity at 45°C and pH 8.5. The kinetics of NcmD toward nocamycin F was investigated at 45°C, pH 8.5 in the presence of 2 mM NAD(+). The K(m) and k(cat) values were 131 ± 13 μM and 65 ± 5 min(−1), respectively. In this study, we have characterized NcmD as a dehydrogenase, which is involved in forming the ketone group at the C-10 position of nocamycin F. The results provide new insights to the nocamycin biosynthetic pathway. Frontiers Media S.A. 2020-12-17 /pmc/articles/PMC7773637/ /pubmed/33391238 http://dx.doi.org/10.3389/fmicb.2020.610827 Text en Copyright © 2020 Mo, Zhang, Du and Yang. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Microbiology Mo, Xuhua Zhang, Hui Du, Fengyu Yang, Song Short-Chain Dehydrogenase NcmD Is Responsible for the C-10 Oxidation of Nocamycin F in Nocamycin Biosynthesis |
title | Short-Chain Dehydrogenase NcmD Is Responsible for the C-10 Oxidation of Nocamycin F in Nocamycin Biosynthesis |
title_full | Short-Chain Dehydrogenase NcmD Is Responsible for the C-10 Oxidation of Nocamycin F in Nocamycin Biosynthesis |
title_fullStr | Short-Chain Dehydrogenase NcmD Is Responsible for the C-10 Oxidation of Nocamycin F in Nocamycin Biosynthesis |
title_full_unstemmed | Short-Chain Dehydrogenase NcmD Is Responsible for the C-10 Oxidation of Nocamycin F in Nocamycin Biosynthesis |
title_short | Short-Chain Dehydrogenase NcmD Is Responsible for the C-10 Oxidation of Nocamycin F in Nocamycin Biosynthesis |
title_sort | short-chain dehydrogenase ncmd is responsible for the c-10 oxidation of nocamycin f in nocamycin biosynthesis |
topic | Microbiology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773637/ https://www.ncbi.nlm.nih.gov/pubmed/33391238 http://dx.doi.org/10.3389/fmicb.2020.610827 |
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