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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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Autores principales: Garcia, David E., Keasling, Jay D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2014
Materias:
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.
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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
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_short Kinetics of Phosphomevalonate Kinase from Saccharomyces cerevisiae
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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