Efficient Base-Catalyzed Kemp Elimination in an Engineered Ancestral Enzyme.

Area of Science:

• Protein engineering
• Biochemistry
• Enzyme catalysis

Background:

• Generating enzymes with novel active sites is challenging using modern proteins as scaffolds.
• Ancestral proteins may offer better biophysical properties for de novo engineering due to their inherent evolvability.

Purpose of the Study:

• To engineer an ancestral protein scaffold for de novo enzyme generation.
• To create an efficient catalyst for the Kemp elimination reaction using protein engineering.

Main Methods:

• Utilized a minimalist protein engineering approach based on an ancestral β-lactamase scaffold.
• Incorporated a single function-generating mutation and an additional polypeptide segment.
• Employed focused, low-throughput library screening for enzyme identification.

Main Results:

• Successfully engineered an ancestral β-lactamase to catalyze Kemp elimination with high efficiency.
• Achieved catalytic parameters (kcat/KM ~2·10^5 M^-1 s^-1, kcat ~635 s^-1) comparable to state-of-the-art de novo enzymes.
• Demonstrated the viability of ancestral proteins as scaffolds for generating new enzyme functions.

Conclusions:

• Ancestral proteins are promising scaffolds for de novo enzyme engineering.
• Minimalist design strategies combined with targeted screening can yield highly active engineered enzymes.
• This work provides a foundation for creating novel enzymes with tailored catalytic activities.