TY - JOUR
T1 - The structure of a tetrameric α-carbonic anhydrase from Thermovibrio ammonificans reveals a core formed around intermolecular disulfides that contribute to its thermostability
AU - James, Paul
AU - Isupov, Michail N.
AU - Sayer, Christopher
AU - Saneei, Vahid
AU - Berg, Svein
AU - Lioliou, Maria
AU - Kotlar, Hans Kristian
AU - Littlechild, Jennifer A
PY - 2014/10/8
Y1 - 2014/10/8
N2 - Carbonic anhydrase enzymes catalyse the reversible hydration of carbon dioxide to bicarbonate. A thermophilic Thermovibrio ammonificans α-carbonic anhydrase (TaCA) has been expressed in Escherichia coli and structurally and biochemically characterized. The crystal structure of TaCA has been determined in its native form and in two complexes with bound inhibitors. The tetrameric enzyme is stabilized by a unique core in the centre of the molecule formed by two intersubunit disulfides and a single lysine residue from each monomer that is involved in intersubunit ionic interactions. The structure of this core protects the intersubunit disulfides from reduction, whereas the conserved intrasubunit disulfides are not formed in the reducing environment of the E. coli host cytosol. When oxidized to mimic the environment of the periplasmic space, TaCA has increased thermostability, retaining 90% activity after incubation at 70°C for 1 h, making it a good candidate for industrial carbon-dioxide capture. The reduction of all TaCA cysteines resulted in dissociation of the tetrameric molecule into monomers with lower activity and reduced thermostability. Unlike other characterized α-carbonic anhydrases, TaCA does not display esterase activity towards p-nitrophenyl acetate, which appears to result from the increased rigidity of its protein scaffold.
AB - Carbonic anhydrase enzymes catalyse the reversible hydration of carbon dioxide to bicarbonate. A thermophilic Thermovibrio ammonificans α-carbonic anhydrase (TaCA) has been expressed in Escherichia coli and structurally and biochemically characterized. The crystal structure of TaCA has been determined in its native form and in two complexes with bound inhibitors. The tetrameric enzyme is stabilized by a unique core in the centre of the molecule formed by two intersubunit disulfides and a single lysine residue from each monomer that is involved in intersubunit ionic interactions. The structure of this core protects the intersubunit disulfides from reduction, whereas the conserved intrasubunit disulfides are not formed in the reducing environment of the E. coli host cytosol. When oxidized to mimic the environment of the periplasmic space, TaCA has increased thermostability, retaining 90% activity after incubation at 70°C for 1 h, making it a good candidate for industrial carbon-dioxide capture. The reduction of all TaCA cysteines resulted in dissociation of the tetrameric molecule into monomers with lower activity and reduced thermostability. Unlike other characterized α-carbonic anhydrases, TaCA does not display esterase activity towards p-nitrophenyl acetate, which appears to result from the increased rigidity of its protein scaffold.
KW - Acetazolamide/chemistry
KW - Bacteria/chemistry
KW - Bacterial Proteins/chemistry
KW - Carbon Dioxide/metabolism
KW - Carbonic Anhydrase Inhibitors/chemistry
KW - Carbonic Anhydrases/chemistry
KW - Catalytic Domain
KW - Cloning, Molecular
KW - Crystallography, X-Ray
KW - Disulfides/chemistry
KW - Enzyme Stability
KW - Kinetics
KW - Models, Molecular
KW - Nitrophenols/metabolism
KW - Protein Conformation
KW - Sulfanilamide
KW - Sulfanilamides/chemistry
KW - Temperature
U2 - 10.1107/S1399004714016526
DO - 10.1107/S1399004714016526
M3 - Article
C2 - 25286845
VL - 70
SP - 2607
EP - 2618
JO - Acta Crystallographica Section D: Biological Crystallography
JF - Acta Crystallographica Section D: Biological Crystallography
SN - 0907-4449
IS - 10
ER -