title: Unfolding and refolding of Aspergillus niger PhyB phytase: role of disulfide bridges.
abstract: The role of disulfide bridges in the folding of Aspergillus niger phytase pH 2.5-optimum (PhyB) was investigated using dynamic light scattering (DLS). Guanidinium chloride (GuCl) at 1.0 M unfolded phytase; however, its removal by dialysis refolded the protein. The thiol reagent tris(2-carboxyethyl)phosphine (TCEP) reduces the refolding activity by 68%. The hydrodynamic radius (R(H)) of PhyB phytase decreased from 5.5 to 4.14 nm when the protein was subjected to 1.0 M GuCl concentration. The active homodimer, 183 kDa, was reduced to a 92 kDa monomer. The DLS data taken together with activity measurements could indicate whether refolding took place or not in PhyB phytase. The correlation between molecular mass and the state of unfolding and refolding is a very strong one in fungal phytase belonging to histidine acid phosphatase (HAP). Unlike PhyA phytase, for which sodium chloride treatment boosted the activity at 0.5 M salt concentration, PhyB phytase activity was severely inhibited under identical condition. Thus, PhyA and PhyB phytases are structurally very different, and their chemical environment in the active site and substrate-binding domain may be different to elicit such an opposite reaction to monovalent cations.
authors.affiliation: Commodity Utilization Research Unit, Southern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 1100 Robert E. Lee Boulevard, New Orleans, Louisiana 70124, USA. abul.ullah@ars.usda.gov
authors.forename: Abul H J
authors.initials: AH
authors.lastname: Ullah
authors.forename: Kandan
authors.initials: K
authors.lastname: Sethumadhavan
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Mullaney
mesh_terms: D010832:6-Phytase / Q000737:chemistry*; D001234:Aspergillus niger / Q000201:enzymology*; D001665:Binding Sites; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004220:Disulfides / Q000737:chemistry*; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D017510:Protein Folding
chemical_list: D004220:Disulfides; D010832:6-Phytase; D019791:Guanidine