Arthrobacter D-xylose isomerase: partial proteolysis with thermolysin

Area of Science:

• Biochemistry
• Protein structure and function
• Enzymology

Background:

• Arthrobacter D-xylose isomerase is a tetrameric enzyme crucial for xylose metabolism.
• Understanding protein loop flexibility is key to elucidating enzyme stability and function.
• Partial proteolysis is a method to probe structural dynamics and identify flexible regions.

Purpose of the Study:

• To investigate the flexibility of surface loops in Arthrobacter D-xylose isomerase.
• To determine how loop flexibility influences the enzyme's stability.
• To identify the key domains controlling the thermostability of the enzyme.

Main Methods:

• Partial proteolysis using thermolysin, trypsin, chymotrypsin, and elastase.
• Analysis of protein subunits by mass spectrometry and SDS-PAGE.
• Enzyme activity assays and stability studies in the presence of metal ions and xylitol.

Main Results:

• Thermolysin specifically cleaved the Thr-347-Leu-348 loop, removing C-terminal residues and yielding a 38 kDa tetramer.
• Further proteolysis at high temperatures removed helices 9 and 10, resulting in a 36 kDa tetramer.
• The Thr-347-Leu-348 loop exhibits temperature-dependent flexibility, becoming fully flexible above 34°C, influenced by Ca2+ binding.
• Nicked enzyme forms (38 kDa and 36 kDa tetramers) retained full activity and similar dissociation properties to the native enzyme.
• Thermostability was largely unaffected by C-terminal deletions, indicating the N-terminal domain controls overall stability.

Conclusions:

• The C-terminal surface loops of Arthrobacter D-xylose isomerase are flexible and can be proteolytically removed without compromising enzyme activity or tetramer stability.
• Ca2+ binding at the active site is linked to the flexibility of the Thr-347-Leu-348 loop.
• The N-terminal beta-barrel domain contains the critical 'weak point' that dictates the overall thermostability of the enzyme.