Download MendeleyCalcium-mediated thermostability in the subtilisin superfamily: the crystal structure of Bacillus Ak.1 protease at 1.8 A resolution.
Smith, C.A., Toogood, H.S., Baker, H.M., Daniel, R.M., Baker, E.N.(1999) J Mol Biol 294: 1027-1040
- PubMed: 10588904
- DOI: https://doi.org/10.1006/jmbi.1999.3291
- Primary Citation of Related Structures:
1DBI - PubMed Abstract:
Proteins of the subtilisin superfamily (subtilases) are widely distributed through many living species, where they perform a variety of processing functions. They are also used extensively in industry. In many of these enzymes, bound calcium ions play a key role in protecting against autolysis and thermal denaturation. We have determined the crystal structure of a highly thermostable protease from Bacillus sp. Ak.1 that is strongly stabilized by calcium. The crystal structure, determined at 1.8 A resolution (R=0. 182, Rfree=0.247), reveals the presence of four bound cations, three Ca(2+) and one Na(+). Two of the Ca(2+) binding sites, Ca-1 and Ca-2, correspond to sites also found in thermitase and the mesophilic subtilisins. The third calcium ion, however, is at a novel site that is created by two key amino acid substitutions near Ca-1, and has not been observed in any other subtilase. This site, acting cooperatively with Ca-1, appears to give substantially enhanced thermostability, compared with thermitase. Comparisons with the mesophilic subtilisins also point to the importance of aromatic clusters, reduced hydrophobic surface and constrained N and C termini in enhancing the thermostability of thermitase and Ak.1 protease. The Ak.1 protease also contains an unusual Cys-X-Cys disulfide bridge that modifies the active site cleft geometry.
Organizational Affiliation:
School of Biological Sciences, University of Auckland, Auckland, New Zealand. ca.smith@auckland.ac.nz
