Structural studies on glycerol dehydrogenase from Bacillus stearothermophilus
Structural studies on glycerol dehydrogenase from Bacillus stearothermophilus
The glycerol dehydrogenase (GDH) (E.C. 1.1.1.6) from Bacillus stearothermophilus is a tetrameric (subunit Mr 39 500). Zn2+-dependent enzyme which catalyses the reversible, NAD+-dependent oxidation of glycerol to dihydroxyacetone. Removal of the metal ion renders the enzyme inactive, implying a catalytic role for this ion. The metal depleted form of the enzyme undergoes a conformational change at low temperatures and high pH which can result in the inability to rebind metal ions and lead to dissociation of the subunits.
Chemical modification experiments previously carried out on GDH have identified a number of residues essential to the structural stability and catalytic activity of the enzyme. Site-directed mutagenesis has been used to probe the role of these residues, and others predicted to play a role in tetramer stability. The mutations which lead to reduced affinity for both the coenzyme and the substrate glycerol usually result in an increased tendency of the oligomer to dissociate, suggesting that the active site may be located between the subunits. The monomers are catalytically inactive and unable to bind the coenzyme, although incubation with high concentrations of coenzymes leads to partially regain of catalytic activity and oligomeric status. The application of these techniques has also led to the identification of residues involved in the ligation of the essential Zn2+ ion.
Fluorescence techniques have been used to characterise the enzyme-coenzyme binding reaction and to estimate distances between the NADH binding domain and the two tryptophan residues in each subunit. In combination with circular dichroism spectroscopy, these techniques have also been used to investigate the conformational stability of the enzyme and characterise a potential folding intermediate or "molten-globule" state occurring in the monomeric mutant C208H.
Limited proteolysis of this monomeric mutant has resulted in the identification of several exposed sites. Secondary structure prediction programs have suggested that the sites identified exist as flexible regions, which are often found to be susceptible to proteolysis.
University of Southampton
1998
Drewett, Victoria Louise
(1998)
Structural studies on glycerol dehydrogenase from Bacillus stearothermophilus.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
The glycerol dehydrogenase (GDH) (E.C. 1.1.1.6) from Bacillus stearothermophilus is a tetrameric (subunit Mr 39 500). Zn2+-dependent enzyme which catalyses the reversible, NAD+-dependent oxidation of glycerol to dihydroxyacetone. Removal of the metal ion renders the enzyme inactive, implying a catalytic role for this ion. The metal depleted form of the enzyme undergoes a conformational change at low temperatures and high pH which can result in the inability to rebind metal ions and lead to dissociation of the subunits.
Chemical modification experiments previously carried out on GDH have identified a number of residues essential to the structural stability and catalytic activity of the enzyme. Site-directed mutagenesis has been used to probe the role of these residues, and others predicted to play a role in tetramer stability. The mutations which lead to reduced affinity for both the coenzyme and the substrate glycerol usually result in an increased tendency of the oligomer to dissociate, suggesting that the active site may be located between the subunits. The monomers are catalytically inactive and unable to bind the coenzyme, although incubation with high concentrations of coenzymes leads to partially regain of catalytic activity and oligomeric status. The application of these techniques has also led to the identification of residues involved in the ligation of the essential Zn2+ ion.
Fluorescence techniques have been used to characterise the enzyme-coenzyme binding reaction and to estimate distances between the NADH binding domain and the two tryptophan residues in each subunit. In combination with circular dichroism spectroscopy, these techniques have also been used to investigate the conformational stability of the enzyme and characterise a potential folding intermediate or "molten-globule" state occurring in the monomeric mutant C208H.
Limited proteolysis of this monomeric mutant has resulted in the identification of several exposed sites. Secondary structure prediction programs have suggested that the sites identified exist as flexible regions, which are often found to be susceptible to proteolysis.
This record has no associated files available for download.
More information
Published date: 1998
Identifiers
Local EPrints ID: 463565
URI: http://eprints.soton.ac.uk/id/eprint/463565
PURE UUID: d4bd934b-7e61-4ae0-95c9-156e72b50f96
Catalogue record
Date deposited: 04 Jul 2022 20:53
Last modified: 04 Jul 2022 20:53
Export record
Contributors
Author:
Victoria Louise Drewett
Download statistics
Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.
View more statistics