Nitrilase of Rhodococcus rhodochrous J1. Conversion into the active form by subunit association

Area of Science:

• Biochemistry
• Enzymology
• Microbial Biotechnology

Background:

• Rhodococcus rhodochrous J1 nitrilase exhibits nitrile hydratase activity in whole cells.
• Purified nitrilase shows substrate specificity differences compared to cellular activity.

Purpose of the Study:

• To investigate the discrepancy in substrate specificity between whole cells and purified nitrilase.
• To elucidate the mechanism of acrylonitrile hydrolysis by purified Rhodococcus rhodochrous J1 nitrilase.

Main Methods:

• Enzyme purification and activity assays.
• Substrate specificity testing with acrylonitrile and benzonitrile.
• Light-scattering experiments to study enzyme aggregation.
• Effect of preincubation, salts, and solvents on enzyme activity.

Main Results:

• Purified nitrilase hydrolyzed benzonitrile but not acrylonitrile.
• Acrylonitrile hydrolysis activity was restored by preincubation with benzonitrile.
• Benzonitrile induced the assembly of the inactive 80-kDa homodimer into an active 410-kDa aggregate (proposed decamer).
• Enzyme activation and aggregation were facilitated by dialysis against specific salts and organic solvents, increased temperature, and enzyme concentration.

Conclusions:

• The purified nitrilase requires activation via aggregation, induced by benzonitrile, to hydrolyze acrylonitrile.
• Enzyme assembly into a higher-order structure is crucial for its activity on acrylonitrile.
• This study reveals a unique activation mechanism for nitrilase enzymes, dependent on substrate-induced oligomerization.