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Characterization of CYP125A13, the First Steroid C-27 Monooxygenase from Streptomyces peucetius ATCC27952
The characterization of cytochrome P450 CYP125A13 from Streptomyces peucetius was conducted using cholesterol as the sole substrate. The in vitro enzymatic assay utilizing putidaredoxin and putidaredoxin reductase from Pseudomonas putida revealed that CYP125A13 bound cholesterol and hydroxylated it....
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Korean Society for Microbiology and Biotechnology
2020
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9728343/ https://www.ncbi.nlm.nih.gov/pubmed/32958729 http://dx.doi.org/10.4014/jmb.2007.07004 |
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author | Rimal, Hemraj Subedi, Pradeep Kim, Ki -Hwa Park, Hyun Lee, Jun Hyuck Oh, Tae-Jin |
author_facet | Rimal, Hemraj Subedi, Pradeep Kim, Ki -Hwa Park, Hyun Lee, Jun Hyuck Oh, Tae-Jin |
author_sort | Rimal, Hemraj |
collection | PubMed |
description | The characterization of cytochrome P450 CYP125A13 from Streptomyces peucetius was conducted using cholesterol as the sole substrate. The in vitro enzymatic assay utilizing putidaredoxin and putidaredoxin reductase from Pseudomonas putida revealed that CYP125A13 bound cholesterol and hydroxylated it. The calculated K(D) value, catalytic conversion rates, and K(m) value were 56.92 ± 11.28 μM, 1.95 nmol min(−1) nmol(−1), and 11.3 ± 2.8 μM, respectively. Gas chromatography-mass spectrometry (GC-MS) analysis showed that carbon 27 of the cholesterol side-chain was hydroxylated, characterizing CYP125A13 as steroid C27-hydroxylase. The homology modeling and docking results also revealed the binding of cholesterol to the active site, facilitated by the hydrophobic amino acids and position of the C27-methyl group near heme. This orientation was favorable for the hydroxylation of the C27-methyl group, supporting the in vitro analysis. This was the first reported case of the hydroxylation of cholesterol at the C-27 position by Streptomyces P450. This study also established the catalytic function of CYP125A13 and provides a solid basis for further studies related to the catabolic potential of Streptomyces species. |
format | Online Article Text |
id | pubmed-9728343 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Korean Society for Microbiology and Biotechnology |
record_format | MEDLINE/PubMed |
spelling | pubmed-97283432022-12-13 Characterization of CYP125A13, the First Steroid C-27 Monooxygenase from Streptomyces peucetius ATCC27952 Rimal, Hemraj Subedi, Pradeep Kim, Ki -Hwa Park, Hyun Lee, Jun Hyuck Oh, Tae-Jin J Microbiol Biotechnol Research article The characterization of cytochrome P450 CYP125A13 from Streptomyces peucetius was conducted using cholesterol as the sole substrate. The in vitro enzymatic assay utilizing putidaredoxin and putidaredoxin reductase from Pseudomonas putida revealed that CYP125A13 bound cholesterol and hydroxylated it. The calculated K(D) value, catalytic conversion rates, and K(m) value were 56.92 ± 11.28 μM, 1.95 nmol min(−1) nmol(−1), and 11.3 ± 2.8 μM, respectively. Gas chromatography-mass spectrometry (GC-MS) analysis showed that carbon 27 of the cholesterol side-chain was hydroxylated, characterizing CYP125A13 as steroid C27-hydroxylase. The homology modeling and docking results also revealed the binding of cholesterol to the active site, facilitated by the hydrophobic amino acids and position of the C27-methyl group near heme. This orientation was favorable for the hydroxylation of the C27-methyl group, supporting the in vitro analysis. This was the first reported case of the hydroxylation of cholesterol at the C-27 position by Streptomyces P450. This study also established the catalytic function of CYP125A13 and provides a solid basis for further studies related to the catabolic potential of Streptomyces species. Korean Society for Microbiology and Biotechnology 2020-11-28 2020-09-15 /pmc/articles/PMC9728343/ /pubmed/32958729 http://dx.doi.org/10.4014/jmb.2007.07004 Text en Copyright©2020 by The Korean Society for Microbiology and Biotechnology https://creativecommons.org/licenses/by/4.0/This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Research article Rimal, Hemraj Subedi, Pradeep Kim, Ki -Hwa Park, Hyun Lee, Jun Hyuck Oh, Tae-Jin Characterization of CYP125A13, the First Steroid C-27 Monooxygenase from Streptomyces peucetius ATCC27952 |
title | Characterization of CYP125A13, the First Steroid C-27 Monooxygenase from Streptomyces peucetius ATCC27952 |
title_full | Characterization of CYP125A13, the First Steroid C-27 Monooxygenase from Streptomyces peucetius ATCC27952 |
title_fullStr | Characterization of CYP125A13, the First Steroid C-27 Monooxygenase from Streptomyces peucetius ATCC27952 |
title_full_unstemmed | Characterization of CYP125A13, the First Steroid C-27 Monooxygenase from Streptomyces peucetius ATCC27952 |
title_short | Characterization of CYP125A13, the First Steroid C-27 Monooxygenase from Streptomyces peucetius ATCC27952 |
title_sort | characterization of cyp125a13, the first steroid c-27 monooxygenase from streptomyces peucetius atcc27952 |
topic | Research article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9728343/ https://www.ncbi.nlm.nih.gov/pubmed/32958729 http://dx.doi.org/10.4014/jmb.2007.07004 |
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