Crystal structure of thermitase at 1.4 A resolution

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Thermitase is a subtilisin-type serine proteinase from Thermoactinomyces vulgaris.
• Subtilisins are a well-studied class of proteases with significant industrial and biotechnological applications.
• Understanding the structural basis of thermitase's enhanced stability is crucial for protein engineering.

Purpose of the Study:

• To determine the high-resolution crystal structure of thermitase.
• To elucidate the structural basis for thermitase's enhanced stability compared to other subtilisins.
• To identify key structural differences contributing to thermitase's unique properties.

Main Methods:

• X-ray diffraction at 1.4 A resolution.
• Molecular and isomorphous replacement for structure solution.
• High-resolution refinement using synchrotron radiation and Fuji image plate detector.

Main Results:

• The crystal structure of thermitase was refined to an R factor of 14.9%.
• Thermitase exhibits unique structural features, including an N-terminal region and an additional beta-strand forming a second Ca2+ binding site.
• These structural differences, particularly in Ca2+ binding and increased ionic/aromatic interactions, correlate with enhanced stability.

Conclusions:

• Thermitase shares overall structural similarity with other subtilisins but possesses distinct features.
• The N-terminal region and the second Ca2+ binding site are critical for thermitase's stability.
• These findings offer valuable insights into the structure-stability relationships of serine proteinases.