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 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 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 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.
Joint BioEnergy Institute, Emeryville, California, United States of America ; Department of Chemistry, University of California, Berkeley, California, United States of America ; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America.
David E
DE
Garcia
Joint BioEnergy Institute, Emeryville, California, United States of America ; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America ; Department of Chemical & Biomolecular Engineering, University of California, Berkeley, California, United States of America ; Department of Bioengineering, University of California, Berkeley, California, United States of America.
Jay D
JD
Keasling
D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D053898:Biosynthetic Pathways / Q000502:physiology*; D003001:Cloning, Molecular; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000254:growth & development; D013696:Temperature
D000255:Adenosine Triphosphate; D017856:Phosphotransferases (Phosphate Group Acceptor); C022371:phosphomevalonate kinase