Conformational Tinkering Drives Evolution of a Promiscuous Activity through Indirect Mutational Effects

Area of Science:

• Evolutionary biochemistry and biophysics
• Enzyme engineering
• Molecular evolution

Background:

• Understanding how distant mutations affect enzyme function is key in evolutionary biochemistry.
• The metallo-β-lactamase (MBL) superfamily includes enzymes like bacterial lactonase (AiiA).

Purpose of the Study:

• To dissect the molecular basis of optimized phosphotriesterase activity in AiiA through laboratory evolution.
• To investigate the role of remote mutations in altering enzyme function and active site dynamics.

Main Methods:

• Laboratory evolution of AiiA for enhanced phosphotriesterase activity.
• Biochemical, structural, genetic, and computational analyses.
• Comparative mutational scanning (alanine mutagenesis).

Main Results:

• A 1000-fold increase in phosphotriesterase activity was achieved.
• Peripheral mutations induced conformational changes, optimizing substrate binding.
• A second-shell mutation displaced Phe68, enabling new interactions with paraoxon substrate.

Conclusions:

• Remote, or second-shell, mutations significantly impact enzyme catalysis through indirect conformational effects.
• Epistatic relationships between active site and remote residues are critical for enzyme evolution.
• This study quantifies the long-range effects of mutations on enzyme function.