Expression and purification of Arg196 and Lys272 mutants of mevalonate kinase from Methanococcus jannaschii

Xiusheng Chu, Xiaojun Liu, Mabel Yau, Yun Chung Leung, Ding Li

Research output: Journal article publicationJournal articleAcademic researchpeer-review

6 Citations (Scopus)

Abstract

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.
Original languageEnglish
Pages (from-to)210-218
Number of pages9
JournalProtein Expression and Purification
Volume30
Issue number2
DOIs
Publication statusPublished - 1 Aug 2003

Keywords

  • Isoprenoid biosynthesis
  • Methanococcus jannaschii
  • Mevalonate kinase
  • Site-directed mutagenesis

ASJC Scopus subject areas

  • Biotechnology

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