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

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' [gamma-thio] triphosphate (ATP-gamma-S), adenosine 5' [beta,gamma-imido] triphosphate (ATP-beta,gamma-NH), adenosine 5' [beta-thio] diphosphate (ADP-beta-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% beta-helices and 26% alpha-sheets. The unfolding equilibrium of rBmG6PD with GdmCl/urea showed the triphasic unfolding pattern along with the highly stable intermediate obtained by GdmCl.