Expression and purification of Arg196 and Lys272 mutants of mevalonate kinase from Methanococcus jannaschii.
In microorganisms and plants, mevalonate kinase is involved in the biosynthesis of isoprenoid derivatives, one of the largest groups of natural products. We subcloned the gene of mevalonate kinase from Methanococcus jannaschii into a bacterial expression vector pLM1 with six continuous histidine codons attached to the 5' end of the gene. A variety of mutant expression plasmids including pMMK(R196K), pMMK(R196Q), pMMK(R196V), pMMK(K272R), and pMMK(K272A) have been constructed using site-directed mutagenesis. The wild-type protein and mutants were overexpressed and purified with a nickel HiTrap chelating metal affinity column to homogeneity. CD spectroscopy of wild-type protein and mutants indicates that none of the above mutations induces significant secondary structural changes. The results from kinetic studies showed that Arg196 is an essential residue for the function of the enzyme. Kinetic studies of Lys272 mutants indicate that salt bridge Lys272-Glu14 plays an important role in maintaining the active site microenvironment that is essential for catalytic activity of the enzyme.
Chu
Xiusheng
X
Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong SAR, PR China.
Liu
Xiaojun
X
Yau
Mabel
M
Leung
Yun-Chung
YC
Li
Ding
D
D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008239:Lysine / Q000235:genetics*; D017017:Methanococcus / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment
D011994:Recombinant Proteins; D001120:Arginine; D017853:Phosphotransferases (Alcohol Group Acceptor); C021796:mevalonate kinase; D008239:Lysine