Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Mo, Xuhua, Zhang, Hui, Du, Fengyu, Yang, Song
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2020
Materias:
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
_version_ 1783630086390415360
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
work_keys_str_mv AT moxuhua shortchaindehydrogenasencmdisresponsibleforthec10oxidationofnocamycinfinnocamycinbiosynthesis
AT zhanghui shortchaindehydrogenasencmdisresponsibleforthec10oxidationofnocamycinfinnocamycinbiosynthesis
AT dufengyu shortchaindehydrogenasencmdisresponsibleforthec10oxidationofnocamycinfinnocamycinbiosynthesis
AT yangsong shortchaindehydrogenasencmdisresponsibleforthec10oxidationofnocamycinfinnocamycinbiosynthesis