Protein engineering using mutability landscapes: Controlling site-selectivity of P450-catalyzed steroid hydroxylation

Area of Science:

• Enzyme engineering
• Protein engineering
• Biocatalysis

Background:

• Directed evolution and rational design are key for enzyme engineering in synthetic chemistry and biotechnology.
• Stereoselectivity is critical for synthesizing valuable chemical and pharmaceutical compounds.
• Directed evolution shows limitations in stereoselectivity and involves laborious screening, motivating new methods.

Purpose of the Study:

• To apply mutability landscaping for improving P450BM3 enzyme.
• To enhance regio-/stereoselectivity and activity in steroid hydroxylation.
• To develop efficient protein engineering with minimal screening.

Main Methods:

• Utilized mutability landscaping, an efficient protein engineering approach.
• Focused on improving P450BM3 for steroid hydroxylation reactions.
• Employed small mutagenesis libraries to reduce screening efforts.

Main Results:

• Successfully improved regio-/stereoselectivity of P450BM3.
• Enhanced the catalytic activity of the engineered enzyme.
• Demonstrated the effectiveness of mutability landscaping in enzyme optimization.

Conclusions:

• Mutability landscaping offers an efficient alternative to traditional methods for enzyme engineering.
• This approach successfully enhances enzyme performance, specifically for P450BM3 in steroid hydroxylation.
• The method allows for significant improvements with reduced screening burden.