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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Public Library of Science
2014
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| Online Access: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3903622/ |
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pubmed-39036222014-01-28 Kinetics of Phosphomevalonate Kinase from Saccharomyces cerevisiae Garcia, David E. Keasling, Jay D. 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 Ni2+ column. The KM 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 KM of the mevalonate-5-phosphate binding site was determined to be 885 µM at 30°C and 880 µM at 37°C. The Vmax 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 Mg2+ 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. |
| repository_type |
Open Access Journal |
| institution_category |
Foreign Institution |
| institution |
US National Center for Biotechnology Information |
| building |
NCBI PubMed |
| collection |
Online Access |
| language |
English |
| format |
Online |
| author |
Garcia, David E. Keasling, Jay D. |
| spellingShingle |
Garcia, David E. Keasling, Jay D. Kinetics of Phosphomevalonate Kinase from Saccharomyces cerevisiae |
| author_facet |
Garcia, David E. Keasling, Jay D. |
| author_sort |
Garcia, David E. |
| title |
Kinetics of Phosphomevalonate Kinase from Saccharomyces cerevisiae
|
| title_short |
Kinetics of Phosphomevalonate Kinase from Saccharomyces cerevisiae
|
| title_full |
Kinetics of Phosphomevalonate Kinase from Saccharomyces cerevisiae
|
| title_fullStr |
Kinetics of Phosphomevalonate Kinase from Saccharomyces cerevisiae
|
| title_full_unstemmed |
Kinetics of Phosphomevalonate Kinase from Saccharomyces cerevisiae
|
| title_sort |
kinetics of phosphomevalonate kinase from saccharomyces cerevisiae |
| 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 Ni2+ column. The KM 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 KM of the mevalonate-5-phosphate binding site was determined to be 885 µM at 30°C and 880 µM at 37°C. The Vmax 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 Mg2+ 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. |
| publisher |
Public Library of Science |
| publishDate |
2014 |
| url |
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3903622/ |
| _version_ |
1612051768084004864 |