Abstract
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.
Highlights
The mevalonate pathway is an important conduit for the production of crucial metabolites with a wide array of functions, including terpenoids [1,2], hormones and steroids [3]
Industrial-scale corn-based ethanol production is already a reality in the energy market, ethanol is a less than desirable biofuel because does it divert crops from the food supply, it is not compatible with our current distribution infrastructure or vehicle fleet [10]. Whether these fuel alternatives are five-carbon alcohols derived from the mevalonate pathway intermediates isopentenyl pyrophosphate and dimethylallyl pyrophosphate [11], or downstream, terpene-based molecules like bisabolene [8], further improvement of titers may be realized through a more robust understanding of the enzymes in the mevalonate pathway and the ways in which those enzymes are regulated by metabolic intermediates
Because mevalonate kinase (MK)—a phosphotransferase that acts on mevalonate and ATP to yield mevalonate-5-phosphate—and phosphomevalonate kinase (PMK)—a phosphotransferase that acts on mevalonate-5-phosphate and ATP to yield mevalonate-5-diphosphate—both require ATP to function and downstream prenyl phosphates might act as general ATP binding site inhibitors, PMK was identified as another potential source of pathway regulation
Summary
The mevalonate pathway is an important conduit for the production of crucial metabolites with a wide array of functions, including terpenoids [1,2], hormones and steroids [3]. Proteomics data has previously shown that the fourth and fifth enzymes in the pathway—mevalonate kinase (MK) and phosphomevalonate kinase (PMK), respectively—are expressed at relatively low levels and may be targets for increasing overall isoprenoid production [12,13].
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