Enzymatic Plasticity Inspired by the Diterpene Cyclase CotB2.

Area of Science:

• Biochemistry
• Enzymology
• Computational Biology

Background:

• Enzymatic plasticity, the ability of an enzyme to alter its function, is crucial for redesigning enzyme activity.
• Bacterial diterpene cyclase CotB2 exemplifies this plasticity, with native forms performing specific reactions and mutants exhibiting diverse catalytic functions.

Purpose of the Study:

• To elucidate the biocyclization mechanisms of the CotB2 enzyme.
• To identify the key regulatory factors governing CotB2's enzymatic plasticity and catalytic selectivity.
• To explore the relationship between active site features and product diversity in diterpene cyclases.

Main Methods:

• Multiscale simulations were employed to investigate the reaction pathways.
• Analysis of local electrostatic stabilization effects from active site residues.
• Examination of global active site features such as pocket contour and hydrophobicity.

Main Results:

• The study illuminated the detailed biocyclization mechanism of the linear substrate to cyclooctat-9-en-7-ol.
• Local electrostatic stabilization by aromatic/polar residues and global active site features (contour, hydrophobicity) were identified as major regulators of CotB2 plasticity.
• Comparative studies revealed a correlation between active site pocket plasticity and product diversity across different diterpene cyclases.

Conclusions:

• The findings provide insights into the catalytic mechanisms and regulatory principles of enzymatic plasticity in CotB2.
• The identified regulatory factors offer a basis for predicting enzyme products and rationally reengineering diterpene cyclases for novel functions.