title: Biochemical characterization of recombinant mevalonate kinase from Bacopa monniera.
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) 49 h. 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 Mg(2+), having Km and Vmax 331.9 μM and 719.1 pKat μg(-1), respectively. The values of kcat and kcat/Km for RS-mevalonate were determined to be 143.82 s(-1) and 0.43332 M(-1) s(-1) and kcat and kcat/Km values for ATP were found 150.9 s(-1) and 1.023 M(-1) s(-1). The metal ion studies suggested that BmMK is a metal dependent enzyme and highly active in the presence of MgCl2.
authors.affiliation: Plant Tissue Culture Division, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, 411008, Maharashtra, India.
authors.forename: Uma
authors.initials: U
authors.lastname: Kumari
authors.affiliation: Plant Tissue Culture Division, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, 411008, Maharashtra, India.
authors.forename: Rishi K
authors.initials: RK
authors.lastname: Vishwakarma
authors.affiliation: Plant Tissue Culture Division, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, 411008, Maharashtra, India.
authors.forename: Prashant
authors.initials: P
authors.lastname: Sonawane
authors.affiliation: Plant Tissue Culture Division, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, 411008, Maharashtra, India.
authors.forename: Shakeel
authors.initials: S
authors.lastname: Abbassi
authors.affiliation: Plant Tissue Culture Division, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, 411008, Maharashtra, India. Electronic address: bm.khan@ncl.res.in.
authors.forename: Bashir M
authors.initials: BM
authors.lastname: Khan
mesh_terms: D000595:Amino Acid Sequence; D034681:Bacopa / Q000737:chemistry* / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004789:Enzyme Activation; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000737:chemistry; D007700:Kinetics; D008670:Metals / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010802:Phylogeny; D011994:Recombinant Proteins; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013696:Temperature
chemical_list: D018076:DNA, Complementary; D007477:Ions; D008670:Metals; D011994:Recombinant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C021796:mevalonate kinase
keywords: Bacopa monniera; Enzyme kinetics; Mevalonate kinase; Stability