Identification of a calcium binding site in Staphylococcus hyicus lipase: Generation of calcium-independent variants

In this study we have identified the presence of a high-affinity binding si te for calcium in the lipase from Staphylococcus hyicus. By means of isothe rmal titration calorimetry we showed that the enzyme binds one calcium per molecule of enzyme with a dissociation constant of 55 mu M. The residual ac tivity of the apoenzyme compared to the activity in the presence of calcium ions varies from 65% at 10 degrees C to nearly zero at 40 degrees C. On th e basis of primary sequence alignment with ether staphylococcal lipases and the lipases from Bacillus thermocatenulatus and from Pseudomonas glumae in combination with site-directed mutagenesis, aspartates 354 and 357 could b e identified as calcium ligands. Kinetic measurements with the D357E varian t showed that replacement of Asp357 by a glutamate decreased the affinity f or calcium ions 30-fold. Introduction of a lysine, an asparagine, or an ala nine at position 357 and of a lysine or an asparagine at position 354 resul ted in calcium-independent variants. Isothermal titration calorimetry confi rmed the loss of calcium binding. Although the D357K, D357N, and D357A vari ants did not bind calcium, at room temperature they were nearly as active a s wild-type lipase in the presence of calcium, but at elevated temperatures these calcium-independent lipases showed a reduced activity. Over the whol e temperature range the activities of the D354K and D354N variants are sign ificantly lower than wild-type enzyme in the presence of calcium and are co mparable to the activity of the wild-type apoenzyme. Our results show that binding of calcium is important for the structural stabilization of staphyl ococcal lipases land possibly other lipases) and that it is possible to eng ineer calcium-independent variants on the basis of limited structural homol ogy with another lipase.