Boron Designer Enzyme with a Hybrid Catalytic Dyad

Area of Science:

• Biochemistry
• Synthetic Biology
• Enzyme Engineering

Background:

• Genetically encoded noncanonical amino acids enable novel enzyme activation modes.
• Enzyme catalysis is enhanced by interactions between amino acids, mimicking natural catalytic dyads/triads.

Purpose of the Study:

• To engineer a novel enzyme for kinetic resolution of α-hydroxythioesters using a boron-containing amino acid.
• To investigate the role of adjacent residues in the catalytic activity of designer enzymes.

Main Methods:

• Incorporation of a boron-containing amino acid into the RamR protein scaffold.
• Kinetic resolution of α-hydroxythioesters.
• Analysis using high-resolution mass spectrometry, ¹¹B NMR spectroscopy, and crystal structure analysis.
• Site-directed mutagenesis to optimize enzyme activity.

Main Results:

• A designer enzyme was created capable of resolving α-hydroxythioesters with good selectivity.
• A lysine residue was identified as crucial for catalytic activity, forming a hybrid catalytic dyad with the boronic acid residue.
• Structural and spectroscopic analyses elucidated the enzyme's catalytic mechanism.
• Mutagenesis yielded a variant with a 2-fold improvement in catalytic activity and selectivity.

Conclusions:

• Designer enzymes incorporating noncanonical amino acids can achieve efficient catalysis.
• Hybrid catalytic dyads formed by noncanonical amino acids and natural residues enhance enzyme function.
• This engineered enzyme provides a powerful tool for the kinetic resolution of α-hydroxythioesters.