TY - JOUR
T1 - Biochemical characterization of recombinant mevalonate kinase from Bacopa monniera
AU - Kumari, Uma
AU - Vishwakarma, Rishi K.
AU - Sonawane, Prashant
AU - Abbassi, Shakeel
AU - Khan, Bashir M.
N1 - Funding Information:
This work was supported by Council of Scientific and Industrial Research -Network Project (CSIR-NWP), New Delhi, India. Authors are thankful to Manas Ranjan Sahoo for his help in western blotting. Parth Patel is duly acknowledged for helping in mathematical calculations. Uma Kumari is thankful to Department of Science and Technology (DST) , New Delhi, India for providing fellowship.
Publisher Copyright:
© 2014 Elsevier B.V.
PY - 2015/1/1
Y1 - 2015/1/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84908032029&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84908032029&partnerID=8YFLogxK
U2 - 10.1016/j.ijbiomac.2014.09.030
DO - 10.1016/j.ijbiomac.2014.09.030
M3 - Article
C2 - 25281875
AN - SCOPUS:84908032029
SN - 0141-8130
VL - 72
SP - 776
EP - 783
JO - International Journal of Biological Macromolecules
JF - International Journal of Biological Macromolecules
ER -