title: NADP⁺ binding effects tryptophan accessibility, folding and stability of recombinant B. malayi G6PD.
abstract: Brugia malayi Glucose 6-phosphate dehydrogenase apoenzyme (BmG6PD) was expressed and purified by affinity chromatography to study the differences in kinetic properties of enzyme and the effect of the cofactor NADP(+) binding on enzyme stability. The presence of cofactor NADP(+) influenced the tertiary structure of enzyme due to significant differences in the tryptophan microenvironment. However, NADP(+) binding have no effect on secondary structure of the enzyme. Quenching with acrylamide indicated that two or more tryptophan residues became accessible upon cofactor binding. Unfolding and cross linking study of BmG6PD showed that NADP(+) stabilized the protein in presence of high concentration of urea/GdmCl. A homology model of BmG6PD constructed using human G6PD (PDB id: 2BH9) as a template indicated 34% α-helix, 19% β-sheet and 47% random coil conformations in the predicted model of the enzyme. In the predicted model binding of NADP(+) to BmG6PD was less tight with the structural sites (-10.96 kJ/mol binding score) as compared with the coenzyme site (-15.47 kJ/mol binding score).
authors.affiliation: Division of Biochemistry, CSIR-Central Drug Research Institute, Sector 10, Jankipuram extension, Sitapur Road, Lucknow 226031, Uttar Pradesh, India.
authors.forename: Anita
authors.initials: A
authors.lastname: Verma
authors.affiliation: Division of Molecular and Structural Biology, CSIR-Central Drug Research Institute, Sector 10, Jankipuram extension, Sitapur Road, Lucknow 226031, Uttar Pradesh, India.
authors.forename: Sharat
authors.initials: S
authors.lastname: Chandra
authors.affiliation: Division of Biochemistry, CSIR-Central Drug Research Institute, Sector 10, Jankipuram extension, Sitapur Road, Lucknow 226031, Uttar Pradesh, India.
authors.forename: Manish Kumar
authors.initials: MK
authors.lastname: Suthar
authors.affiliation: Division of Biochemistry, CSIR-Central Drug Research Institute, Sector 10, Jankipuram extension, Sitapur Road, Lucknow 226031, Uttar Pradesh, India.
authors.forename: Pawan Kumar
authors.initials: PK
authors.lastname: Doharey
authors.affiliation: Division of Molecular and Structural Biology, CSIR-Central Drug Research Institute, Sector 10, Jankipuram extension, Sitapur Road, Lucknow 226031, Uttar Pradesh, India.
authors.forename: Mohammad Imran
authors.initials: MI
authors.lastname: Siddiqi
authors.affiliation: Division of Biochemistry, CSIR-Central Drug Research Institute, Sector 10, Jankipuram extension, Sitapur Road, Lucknow 226031, Uttar Pradesh, India. Electronic address: jkscdri@yahoo.com.
authors.forename: Jitendra Kumar
authors.initials: JK
authors.lastname: Saxena
mesh_terms: D000818:Animals; D017178:Brugia malayi / Q000201:enzymology; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008968:Molecular Conformation; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D009249:NADP / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D055550:Protein Stability; D058767:Protein Unfolding; D011994:Recombinant Proteins; D013057:Spectrum Analysis / Q000379:methods; D014364:Tryptophan / Q000737:chemistry* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D009249:NADP; D014364:Tryptophan; D005954:Glucosephosphate Dehydrogenase
keywords: Brugia malayi G6PD; Dissociation constant; Homology model