title: NMR characterization of a 264-residue hyperthermostable endo-beta-1,3-glucanase.
abstract: Insight into the hyperthermostable endo-beta-1,3-glucanase pfLamA from Pyrococcus furiosus is obtained by using NMR spectroscopy. pfLamA functions optimally at 104 degrees C and recently the X-ray structure of pfLamA has been obtained at 20 degrees C, a temperature at which the enzyme is inactive. In this study, near-complete (>99%) NMR assignments are presented of chemical shifts of pfLamA in presence and absence of calcium at 62 degrees C, a temperature at which the enzyme is biologically active. The protein contains calcium and the effects of calcium on the protein are assessed. Calcium binding results in relatively small chemical shift changes in a region distant from the active site of pfLamA and thus causes only minor conformational modifications. Removal of calcium does not significantly alter the denaturation temperature of pfLamA, implying that calcium does not stabilize the enzyme against global unfolding. The data obtained form the basis for elucidation of the molecular origins involved in conformational stability and biological activity of hyperthermophilic endo-beta-1,3-glucanases at extreme temperatures.
authors.affiliation: Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands.
authors.forename: Johannes H
authors.initials: JH
authors.lastname: Ippel
authors.forename: Sotirios
authors.initials: S
authors.lastname: Koutsopoulos
authors.forename: Sanne M
authors.initials: SM
authors.lastname: Nabuurs
authors.forename: Willem J H
authors.initials: WJ
authors.lastname: van Berkel
authors.forename: John
authors.initials: J
authors.lastname: van der Oost
authors.forename: Carlo P M
authors.initials: CP
authors.lastname: van Mierlo
mesh_terms: D002118:Calcium / Q000737:chemistry*; D018360:Crystallography, X-Ray; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000737:chemistry*; D006358:Hot Temperature; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011489:Protein Denaturation; D020027:Pyrococcus furiosus / Q000201:enzymology*
chemical_list: D004693:Glucan Endo-1,3-beta-D-Glucosidase; D002118:Calcium