Beta-Glycosidases are fundamental, widely conserved enzymes. Those from hyperthermophiles exhibit unusual stabilities toward various perturbants. Previous work with homotetrameric beta-glycosidase from hyperthermophilic Sulfolobus solfataricus (Mr 226,760) has shown that addition of 0.05–0.1% SDS was associated with minimal secondary structure perturbations and increased activity. This work addresses the effects of SDS on beta-glycosidase quaternary structure. In 0.1–1% SDS, the enzyme was dimeric, as determined by Ferguson analysis of transverse-gradient polyacrylamide gels. The catalytic activity of the beta-glycosidase dimer in SDS was determined by in-gel assay. A minor decrease of thermal stability in SDS was observed after exposure to temperatures up to 80 °C for 1 h. An analysis of beta-glycosidase crystal structure showed different changes in solvent accessible surface area on going from the tetramer to the two possible dimers (A-C and A-D). Energy minimization and molecular dynamics calculations showed that the A-C dimer, exhibiting the lowest exposed surface area, was more stabilized by a network of polar interactions. The charge distribution around the A-C interface was characterized by a local short range anisotropy, resulting in an unfavorable interaction with SDS. This paper provides a detailed description of an SDS-resistant inter-monomeric interface, which may help understand similar interfaces involved in important biological processes.
Digital Object Identifier (DOI): | http://dx.doi.org/10.1074/jbc.M206761200 |
Codice identificativo ISI: | WOS:000179272000068 |
Codice identificativo Scopus: | 2-s2.0-0346461016 |
Handle: | http://hdl.handle.net/11695/3716 |
Abstract: | Beta-Glycosidases are fundamental, widely conserved enzymes. Those from hyperthermophiles exhibit unusual stabilities toward various perturbants. Previous work with homotetrameric beta-glycosidase from hyperthermophilic Sulfolobus solfataricus (Mr 226,760) has shown that addition of 0.05–0.1% SDS was associated with minimal secondary structure perturbations and increased activity. This work addresses the effects of SDS on beta-glycosidase quaternary structure. In 0.1–1% SDS, the enzyme was dimeric, as determined by Ferguson analysis of transverse-gradient polyacrylamide gels. The catalytic activity of the beta-glycosidase dimer in SDS was determined by in-gel assay. A minor decrease of thermal stability in SDS was observed after exposure to temperatures up to 80 °C for 1 h. An analysis of beta-glycosidase crystal structure showed different changes in solvent accessible surface area on going from the tetramer to the two possible dimers (A-C and A-D). Energy minimization and molecular dynamics calculations showed that the A-C dimer, exhibiting the lowest exposed surface area, was more stabilized by a network of polar interactions. The charge distribution around the A-C interface was characterized by a local short range anisotropy, resulting in an unfavorable interaction with SDS. This paper provides a detailed description of an SDS-resistant inter-monomeric interface, which may help understand similar interfaces involved in important biological processes. |
Titolo: | SDS-resistant active and thermostable dimers are obtained from the dissociation of homotetrameric beta-glycosidase from hyperthermophilic Sulfolobus solfataricus in SDS. Stabilizing role of the A-C intermonomeric interface |
Autori: | |
Appare nelle tipologie: | 1.1 Articolo in rivista |