Biochemical characterization of recombinant mevalonate kinase from Bacopa monniera

Uma Kumari, Rishi K. Vishwakarma, Prashant Sonawane, Shakeel Abbassi, Bashir M. Khan

Research output: Contribution to journalArticlepeer-review

Abstract

Mevalonate kinase (MK; ATP: mevalonate 5-phosphotransferase; EC 2.7.1.36) plays a key role in isoprenoid biosynthetic pathway in plants. MK catalyzes the phosphorylation of mevalonate to form mevalonate-5-phosphate. The recombinant BmMK was cloned and over-expressed in E. coli BL21 (DE3), and purified to homogeneity by affinity chromatography followed by gel filtration. Optimum pH and temperature for forward reaction was found to be 7.0 and 30°C, respectively. The enzyme was most stable at pH 8 at 25°C with deactivation rate constant (Kd*) 1.398×10-4 and half life (t1/2) 49h. pH activity profile of BmMK indicates the involvement of carboxylate ion, histidine, lysine, arginine or aspartic acid at the active site of enzyme. Activity of recombinant BmMK was confirmed by phosphorylation of RS-mevalonate in the presence of Mg2+, having Km and Vmax 331.9μM and 719.1pKatμg-1, respectively. The values of kcat and kcat/Km for RS-mevalonate were determined to be 143.82s-1 and 0.43332M-1s-1 and kcat and kcat/Km values for ATP were found 150.9s-1 and 1.023M-1s-1. The metal ion studies suggested that BmMK is a metal dependent enzyme and highly active in the presence of MgCl2.

Original languageEnglish (US)
Pages (from-to)776-783
Number of pages8
JournalInternational Journal of Biological Macromolecules
Volume72
DOIs
StatePublished - Jan 1 2015

All Science Journal Classification (ASJC) codes

  • Structural Biology
  • Biochemistry
  • Molecular Biology
  • Economics and Econometrics
  • Energy(all)

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