title: A rationally designed monomeric variant of anthranilate phosphoribosyltransferase from Sulfolobus solfataricus is as active as the dimeric wild-type enzyme but less thermostable.
abstract: The anthranilate phosphoribosyltransferase from Sulfolobus solfataricus (ssAnPRT) forms a homodimer with a hydrophobic subunit interface. To elucidate the role of oligomerisation for catalytic activity and thermal stability of the enzyme, we loosened the dimer by replacing two apolar interface residues with negatively charged residues (mutations I36E and M47D). The purified double mutant I36E+M47D formed a monomer with wild-type catalytic activity but reduced thermal stability. The single mutants I36E and M47D were present in a monomer-dimer equilibrium with dissociation constants of about 1 microM and 20 microM, respectively, which were calculated from the concentration-dependence of their heat inactivation kinetics. The monomeric form of M47D, which is populated at low subunit concentrations, was as thermolabile as monomeric I36E+M47D. Likewise, the dimeric form of I36E, which was populated at high subunit concentrations, was as thermostable as dimeric wild-type ssAnPRT. These findings show that the increased stability of wild-type ssAnPRT compared to the I36E+M47D double mutant is not caused by the amino acid exchanges per se but by the higher intrinsic stability of the dimer compared to the monomer. In accordance with the negligible effect of the mutations on catalytic activity and stability, the X-ray structure of M47D contains only minor local perturbations at the dimer interface. We conclude that the monomeric double mutant resembles the individual wild-type subunits, and that ssAnPRT is a dimer for stability but not for activity reasons.
authors.affiliation: Institute of Biophysics und Physical Biochemistry, University of Regensburg, Universitätsstrasse 31, D-93053 Regensburg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Schwab
authors.forename: Darko
authors.initials: D
authors.lastname: Skegro
authors.forename: Olga
authors.initials: O
authors.lastname: Mayans
authors.forename: Reinhard
authors.initials: R
authors.lastname: Sterner
mesh_terms: D000877:Anthranilate Phosphoribosyltransferase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002152:Calorimetry, Differential Scanning; D002384:Catalysis; D018360:Crystallography, X-Ray; D019281:Dimerization; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D014644:Genetic Variation; D006358:Hot Temperature; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D010957:Plasmids; D017433:Protein Structure, Secondary; D013379:Substrate Specificity; D048229:Sulfolobus solfataricus / Q000201:enzymology*
chemical_list: D000877:Anthranilate Phosphoribosyltransferase