Overexpression, purification, and characterization of the thermostable mevalonate kinase from Methanococcus jannaschii.
We report here the first overexpression and characterization of a thermostable mevalonate kinase from an archae, Methanococcus jannaschii, a strict anaerobe, which produces methane and grows at pressure of 200 atm and an optimum temperature near 85 degrees C. PCR-derived DNA fragments containing the structural gene for mevalonate kinase were cloned into an expression vector, pET28a, to form pETMVK. The mevalonate kinase was overexpressed from Escherichia coli pETMVK/BL21(DE3) (15-20% of total soluble protein) when induced with isopropyl beta-d-thiogalactopyranoside. The protein was purified by heat treatment (to denature E. coli proteins), followed by metal-affinity chromatography on Talon metal-affinity resin column. The purified protein had a dimeric structure composed of identical subunits, and the M(r) of the enzyme determined by gel chromatography was 68K. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the subunit M(r) was 36, 000. The pI for mevalonate kinase was 7.8. The Michaelis constant (K(m)) for (RS)-mevalonate was 68.5 microM and was 92 microM for ATP. The V(max) was 387 units mg(-1). The optimal temperature for mevalonate kinase activity was 70-75 degrees C.
Huang
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Department of Biochemistry and Cell Biology, Rice University, 6100 Main Street, Houston, Texas 77005-1892, USA.
Scott
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Bennett
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D000595:Amino Acid Sequence; D001483:Base Sequence; D017124:Conserved Sequence; D017931:DNA Primers / Q000235:genetics; D019281:Dimerization; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D019766:Genes, Archaeal; D005822:Genetic Vectors; D006358:Hot Temperature; D007526:Isoelectric Point; D007700:Kinetics; D017017:Methanococcus / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D011994:Recombinant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C021796:mevalonate kinase