title: Specific potassium binding stabilizes pI258 arsenate reductase from Staphylococcus aureus.
abstract: Arsenate reductase (ArsC) from Staphylococcus aureus plasmid pI258 catalyzes the reduction of arsenate to arsenite and plays a role in bacterial heavy metal resistance. The high resolution x-ray structure of ArsC reveals the atomic details of the K+ binding site situated next to the catalytic P-loop structural motif of this redox enzyme. A full thermodynamic study of the binding characteristics of a series of monovalent cations (Li+, Na+, K+, Rb+, and Cs+) and their influence on the thermal stability of ArsC was performed with isothermal titration calorimetry, circular dichroism spectroscopy, and differential scanning calorimetry. Potassium has the largest affinity with a Ka of 3.8 x 10(3) m(-1), and the effectiveness of stabilization of ArsC by monovalent cations follows the binding affinity order: K+ > Rb+ > Cs+ > Na+ > Li+. A mutagenesis study on the K+ binding side chains showed that Asn-13 and Asp-65 are essential for potassium binding, but the impact on the stability of ArsC was the most extreme when mutating Ser-36. Additionally, the thermal stabilization by K+ is significantly reduced in the case of the ArsC E21A mutant, showing the importance of a Glu-21-coordinated water molecule in its contact with K+. Although potassium is not essential for catalysis, in its presence the kcat/KM increases with a factor of 5. Altogether, the interaction of K+ with specific residues in ArsC is an enthalpydriven process that stabilizes ArsC and increases the specific activity of this redox enzyme.
authors.affiliation: Department Ultrastructure, Vlaams interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Pleinlaan 2, Belgium. nina.lah@uni-lj.si
authors.forename: Nina
authors.initials: N
authors.lastname: Lah
authors.forename: Jurij
authors.initials: J
authors.lastname: Lah
authors.forename: Ingrid
authors.initials: I
authors.lastname: Zegers
authors.forename: Lode
authors.initials: L
authors.lastname: Wyns
authors.forename: Joris
authors.initials: J
authors.lastname: Messens
mesh_terms: D053501:Arsenite Transporting ATPases; D002151:Calorimetry; D002942:Circular Dichroism; D004795:Enzyme Stability; D016623:Ion Pumps / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D009097:Multienzyme Complexes / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011188:Potassium; D011485:Protein Binding; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics
chemical_list: D016623:Ion Pumps; D009097:Multienzyme Complexes; D053501:Arsenite Transporting ATPases; D011188:Potassium