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Molecular cloning and characterization of glucose-6-phosphate dehydrogenase from Brugia malayi

Published online by Cambridge University Press:  18 March 2013

ANITA VERMA
Affiliation:
Division of Biochemistry, CSIR-Central Drug Research Institute, Lucknow-226001, Uttar Pradesh, India
MANISH K. SUTHAR
Affiliation:
Division of Biochemistry, CSIR-Central Drug Research Institute, Lucknow-226001, Uttar Pradesh, India
PAWAN K. DOHAREY
Affiliation:
Division of Biochemistry, CSIR-Central Drug Research Institute, Lucknow-226001, Uttar Pradesh, India
SMITA GUPTA
Affiliation:
Division of Biochemistry, CSIR-Central Drug Research Institute, Lucknow-226001, Uttar Pradesh, India
SUNITA YADAV
Affiliation:
Division of Biochemistry, CSIR-Central Drug Research Institute, Lucknow-226001, Uttar Pradesh, India
PREM M. S. CHAUHAN
Affiliation:
Division of Medicinal and Process Chemistry, CSIR-Central Drug Research Institute, Lucknow-226001, Uttar Pradesh, India
JITENDRA K. SAXENA*
Affiliation:
Division of Biochemistry, CSIR-Central Drug Research Institute, Lucknow-226001, Uttar Pradesh, India
*
*Corresponding author: Division of Biochemistry, CSIR-Central Drug Research Institute, Lucknow-226001, Uttar Pradesh, India. E-mail: jkscdri@yahoo.com

Summary

Glucose-6-phosphate dehydrogenase (G6PD), a regulatory enzyme of the pentose phosphate pathway from Brugia malayi, was cloned, expressed and biochemically characterized. The Km values for glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (NADP) were 0·25 and 0·014 mm respectively. The rBmG6PD exhibited an optimum pH of 8·5 and temperature, 40 °C. Adenosine 5′ [γ-thio] triphosphate (ATP-γ-S), adenosine 5′ [β,γ-imido] triphosphate (ATP-β,γ-NH), adenosine 5′ [β-thio] diphosphate (ADP-β-S), Na+, K+, Li+ and Cu++ ions were found to be strong inhibitors of rBmG6PD. The rBmG6PD, a tetramer with subunit molecular weight of 75 kDa contains 0·02 mol of SH group per mol of monomer. Blocking the SH group with SH-inhibitors, led to activation of rBmG6PD activity by N-ethylmaleimide. CD analysis indicated that rBmG6PD is composed of 37% α-helices and 26% β-sheets. The unfolding equilibrium of rBmG6PD with GdmCl/urea showed the triphasic unfolding pattern along with the highly stable intermediate obtained by GdmCl.

Type
Research Article
Copyright
Copyright © Cambridge University Press 2013 

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