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Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip
Published on: March 20, 2021
Structure of a switchable subtilisin complexed with a substrate and with the activator azide
Travis Gallagher1, Biao Ruan, Mariya London
1Center for Advanced Research in Biotechnology, 9600 Gudelsky Drive, Rockville, Maryland 20850, USA. gallaghe@umbi.umd.edu
Biochemistry
|September 19, 2009
Summary
Engineered subtilisin protease activity is activated over 3000-fold by azide. Structural studies reveal how anions like azide and fluoride trigger this protease, enabling new biotechnology tools for expression and purification.
Area of Science:
- Biochemistry
- Structural Biology
- Biotechnology
Background:
- Subtilisin protease from Bacillus amyloliquefaciens is a well-studied enzyme.
- Engineered variants can offer novel functionalities for biotechnological applications.
Purpose of the Study:
- To engineer a subtilisin variant with anion-dependent activity for use as an expression-purification tool.
- To elucidate the structural basis of anion-activated proteolysis.
Main Methods:
- Site-directed mutagenesis (D32A, S221A) to create engineered subtilisin variants.
- Enzyme activity assays to quantify anion-dependent activation.
- X-ray crystallography to determine the 1.8 Å resolution structure of the enzyme-substrate complex with azide.
Main Results:
- The D32A mutation confers azide- and fluoride-dependent activity, with over 3000-fold activation by azide.
- Crystal structures reveal the binding of azide adjacent to Ala 32 and a well-ordered engineered substrate.
- Comparison of structures in different crystalline states provides insights into anion binding and substrate interaction.
Conclusions:
- Anion-dependent proteolysis mechanism is a slight modification of the canonical serine protease charge-relay mechanism.
- Engineered subtilisin variants with anion-triggered activity hold promise for developing novel biotechnology tools.
- Structural data provides a foundation for understanding and designing anion-activated proteases.
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