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Kinetics of Phosphomevalonate Kinase from Saccharomyces cerevisiae
The mevalonate-based isoprenoid biosynthetic pathway is responsible for producing cholesterol in humans and is used commercially to produce drugs, chemicals, and fuels. Heterologous expression of this pathway in Escherichia coli has enabled high-level production of the antimalarial drug artemisinin...
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Public Library of Science
2014
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3903622/ https://www.ncbi.nlm.nih.gov/pubmed/24475236 http://dx.doi.org/10.1371/journal.pone.0087112 |
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author | Garcia, David E. Keasling, Jay D. |
author_facet | Garcia, David E. Keasling, Jay D. |
author_sort | Garcia, David E. |
collection | PubMed |
description | The mevalonate-based isoprenoid biosynthetic pathway is responsible for producing cholesterol in humans and is used commercially to produce drugs, chemicals, and fuels. Heterologous expression of this pathway in Escherichia coli has enabled high-level production of the antimalarial drug artemisinin and the proposed biofuel bisabolane. Understanding the kinetics of the enzymes in the biosynthetic pathway is critical to optimize the pathway for high flux. We have characterized the kinetic parameters of phosphomevalonate kinase (PMK, EC 2.7.4.2) from Saccharomyces cerevisiae, a previously unstudied enzyme. An E. coli codon-optimized version of the S. cerevisiae gene was cloned into pET-52b+, then the C-terminal 6X His-tagged protein was expressed in E. coli BL21(DE3) and purified on a Ni(2+) column. The K(M) of the ATP binding site was determined to be 98.3 µM at 30°C, the optimal growth temperature for S. cerevisiae, and 74.3 µM at 37°C, the optimal growth temperature for E. coli. The K(M) of the mevalonate-5-phosphate binding site was determined to be 885 µM at 30°C and 880 µM at 37°C. The V(max) was determined to be 4.51 µmol/min/mg enzyme at 30°C and 5.33 µmol/min/mg enzyme at 37°C. PMK is Mg(2+) dependent, with maximal activity achieved at concentrations of 10 mM or greater. Maximum activity was observed at pH = 7.2. PMK was not found to be substrate inhibited, nor feedback inhibited by FPP at concentrations up to 10 µM FPP. |
format | Online Article Text |
id | pubmed-3903622 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-39036222014-01-28 Kinetics of Phosphomevalonate Kinase from Saccharomyces cerevisiae Garcia, David E. Keasling, Jay D. PLoS One Research Article The mevalonate-based isoprenoid biosynthetic pathway is responsible for producing cholesterol in humans and is used commercially to produce drugs, chemicals, and fuels. Heterologous expression of this pathway in Escherichia coli has enabled high-level production of the antimalarial drug artemisinin and the proposed biofuel bisabolane. Understanding the kinetics of the enzymes in the biosynthetic pathway is critical to optimize the pathway for high flux. We have characterized the kinetic parameters of phosphomevalonate kinase (PMK, EC 2.7.4.2) from Saccharomyces cerevisiae, a previously unstudied enzyme. An E. coli codon-optimized version of the S. cerevisiae gene was cloned into pET-52b+, then the C-terminal 6X His-tagged protein was expressed in E. coli BL21(DE3) and purified on a Ni(2+) column. The K(M) of the ATP binding site was determined to be 98.3 µM at 30°C, the optimal growth temperature for S. cerevisiae, and 74.3 µM at 37°C, the optimal growth temperature for E. coli. The K(M) of the mevalonate-5-phosphate binding site was determined to be 885 µM at 30°C and 880 µM at 37°C. The V(max) was determined to be 4.51 µmol/min/mg enzyme at 30°C and 5.33 µmol/min/mg enzyme at 37°C. PMK is Mg(2+) dependent, with maximal activity achieved at concentrations of 10 mM or greater. Maximum activity was observed at pH = 7.2. PMK was not found to be substrate inhibited, nor feedback inhibited by FPP at concentrations up to 10 µM FPP. Public Library of Science 2014-01-27 /pmc/articles/PMC3903622/ /pubmed/24475236 http://dx.doi.org/10.1371/journal.pone.0087112 Text en © 2014 Garcia, Keasling http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Garcia, David E. Keasling, Jay D. Kinetics of Phosphomevalonate Kinase from Saccharomyces cerevisiae |
title | Kinetics of Phosphomevalonate Kinase from Saccharomyces cerevisiae
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title_full | Kinetics of Phosphomevalonate Kinase from Saccharomyces cerevisiae
|
title_fullStr | Kinetics of Phosphomevalonate Kinase from Saccharomyces cerevisiae
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title_full_unstemmed | Kinetics of Phosphomevalonate Kinase from Saccharomyces cerevisiae
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title_short | Kinetics of Phosphomevalonate Kinase from Saccharomyces cerevisiae
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title_sort | kinetics of phosphomevalonate kinase from saccharomyces cerevisiae |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3903622/ https://www.ncbi.nlm.nih.gov/pubmed/24475236 http://dx.doi.org/10.1371/journal.pone.0087112 |
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