Changes in Active Site Loop Conformation Relate to the Transition toward a Novel Enzymatic Activity

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Enzymatic promiscuity is a key factor in the evolution of new enzyme functions.
• The molecular mechanisms underlying the shift between enzyme activities are not fully understood.

Purpose of the Study:

• To investigate the molecular mechanisms of enzyme activity transition.
• To redesign the active site of lactonase SsoPox for altered catalytic functions.

Main Methods:

• Structure-based design and combinatorial libraries were used to engineer SsoPox variants.
• Crystal structures of engineered variants were determined to analyze active site changes.
• Catalytic activity against phosphotriesters was measured to assess functional shifts.

Main Results:

• Engineered variants showed >1000-fold improvement in phosphotriester catalysis compared to wild-type.
• Mutations significantly altered the active site cavity, primarily through loop rearrangements and conformational changes.
• Some variants exhibited complete loss of original lactonase activity, indicating substantial specificity shifts.

Conclusions:

• Active site loop configuration is critical for lactonase activity.
• Conformational sampling and its directionality may play a role in determining enzyme activity profiles.
• Enzyme redesign through structure-based approaches can effectively modulate enzymatic function and specificity.