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Purification and characterization of guanylate kinase, a nucleoside monophosphate kinase of Brugia malayi

Published online by Cambridge University Press:  20 May 2014

SMITA GUPTA ,
SUNITA YADAV ,
NIDHI SINGH ,
ANITA VERMA ,
IMRAN SIDDIQI  and
JITENDRA K. SAXENA
SMITA GUPTA
Affiliation:
Division of Biochemistry, CSIR-Central Drug Research Institute, Lucknow - 226031, Uttar Pradesh, India
SUNITA YADAV
Affiliation:
Division of Biochemistry, CSIR-Central Drug Research Institute, Lucknow - 226031, Uttar Pradesh, India
NIDHI SINGH
Affiliation:
Division of Molecular and Structural Biology, CSIR-Central Drug Research Institute, Lucknow - 226031, Uttar Pradesh, India
ANITA VERMA
Affiliation:
Division of Biochemistry, CSIR-Central Drug Research Institute, Lucknow - 226031, Uttar Pradesh, India
IMRAN SIDDIQI
Affiliation:
Division of Biochemistry, CSIR-Central Drug Research Institute, Lucknow - 226031, Uttar Pradesh, India
JITENDRA K. SAXENA*
Affiliation:
Division of Biochemistry, CSIR-Central Drug Research Institute, Lucknow - 226031, Uttar Pradesh, India
*
*Corresponding author: Division of Biochemistry Central Drug Research Institute BS10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow - 226031, Uttar Pradesh, India. E-mail: [email protected]
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Summary

Guanylate kinase, a nucleoside monophosphate kinase of Brugia malayi which is involved in reversible transfer of phosphate groups from ATP to GMP, was cloned, expressed and characterized. The native molecular mass of BmGK was found to be 45 kDa as determined by size exclusion chromatography and glutaraldehyde cross-linking which revealed that the protein is homodimer in nature. This is a unique characteristic among known eukaryotic GKs. GMP and ATP served as the most effective phosphate acceptor and donor, respectively. Recombinant BmGK utilized both GMP and dGMP, as substrates showing Km values of 30 and 38 μm, respectively. Free Mg+2 (un-complexed to ATP) and GTP play a regulatory role in catalysis of BmGK. The enzyme showed higher catalytic efficiency as compared with human GK and showed ternary complex (BmGK-GMP-ATP) formation with sequential substrate binding. The secondary structure of BmGK consisted of 45% α-helices, 18% β-sheets as revealed by CD analysis. Homology modelling and docking with GMP revealed conserved substrate binding residues with slight differences. Differences in kinetic properties and oligomerization of BmGK compared with human GK can provide the way for design of parasite-specific inhibitors.

Keywords

Brugia malayiguanylate kinasenucleoside monophosphate kinaseoligomerizationendproduct inhibition
Type
Research Article
Information
Parasitology , Volume 141 , Issue 10 , September 2014 , pp. 1341 - 1352
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Copyright
Copyright © Cambridge University Press 2014 

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