Arthrobacter D-xylose isomerase: protein-engineered subunit interfaces

Area of Science:

• Biochemistry
• Enzymology
• Protein Engineering

Background:

• D-xylose isomerase is a crucial enzyme in carbohydrate metabolism.
• Understanding enzyme stability and activity is vital for industrial applications.
• Protein engineering allows modification of enzyme properties.

Purpose of the Study:

• To investigate the impact of engineered disulfide and salt bridges on Arthrobacter D-xylose isomerase.
• To analyze the effects of specific mutations on enzyme stability and catalytic activity.
• To elucidate the structural and mechanistic basis of D-xylose isomerase inactivation.

Main Methods:

• Site-directed mutagenesis to introduce disulfide and salt bridges.
• Enzyme activity assays across various pH and metal ion conditions.
• Thermostability and urea denaturation studies.
• X-ray crystallography to determine enzyme structure.

Main Results:

• Mutations at A-A* interfaces showed no change in activity or stability.
• A Tyr253 mutation (Y253C) at the A-B* interface reduced thermostability and urea stability.
• X-ray crystallography revealed conformational changes in Glu185 and Asp254 in the Y253C mutant.
• The Y253C mutant exhibited altered kinetic parameters (Km, Vmax) and pH-dependent activity changes with different metal ions.
• Electrostatic repulsion and altered metal ion binding at Site [2] were implicated in the Y253C mutant's reduced activity.

Conclusions:

• Engineered inter-subunit bridges at A-A* interfaces do not significantly alter D-xylose isomerase properties.
• The Tyr253 mutation introduces conformational instability, affecting enzyme thermostability and metal ion coordination.
• Subunit dissociation is not the primary mechanism of thermal inactivation; active site conformational changes initiate unfolding.
• The study provides insights into the structural determinants of D-xylose isomerase stability and catalytic mechanism.