Circularly permuted beta-lactamase from Staphylococcus aureus PC1.

The role that domain flexibility plays in the enzymatic activity of beta-lactamase from Staphylococcus aureus PC1 was investigated by producing two circularly permuted molecules. The C- and N-termini of the wild-type enzyme are adjacent to each other and remote from the active site, which is located between two domains. The polypeptide chain crosses over from one domain to the other twice. For the circularly permuted molecules, the termini were joined by an eight amino acid residue insertion, and new termini were introduced elsewhere. The first construct, termed cp254, was cleaved in a loop remote from the domain interface. The crystal structure of cp254 has been determined and refined at 1.8 A resolution, revealing essentially the same structure as that of the native protein. The activity profile with a representative sample of beta-lactam antibiotics is also very similar to that of wild-type beta-lactamase. The termini of the second circularly permuted mutant, cp228, occur within the second crossover region and therefore may enhance the flexibility of the molecule. Cp228 beta-lactamase shows a large decrease in enzymatic activity toward the sample of beta-lactam antibiotics, with catalytic rates that are 0.5-1% of those of the wild-type enzyme. One exception is the hydrolysis of the third generation cephalosporin, cefotaxime, which is hydrolyzed by the cp228 enzyme 10-fold faster than by wild-type beta-lactamase. Cp228 has not been crystallized. However, the circular dichroism spectra of the two circularly permuted proteins are very similar, indicating that, by analogy to cp254, cp228 adopts a global folded state. Thermal denaturation experiments reveal that cp254 is somewhat less stable than the wild-type enzyme, whereas cp228 is substantially less stable. Together, the data highlight the profound consequences that introducing flexibility at the domain interface has on both enzyme activity and protein stability.