Halogenation of Peptides and Proteins Using Engineered Tryptophan Halogenase Enzymes

Area of Science:

• Biochemistry
• Synthetic Biology
• Protein Engineering

Background:

• Halogenation of bioactive peptides is a key strategy for enhancing molecular functionality.
• Bacterial tryptophan halogenases are known for efficient, regiospecific halogenation of free tryptophan.
• Expanding halogenase substrate scope to peptides and proteins is crucial for regulated post-synthesis modification.

Purpose of the Study:

• To demonstrate in vitro halogenation of peptides using select bacterial halogenase enzymes.
• To identify preferred peptide motifs for enzymatic halogenation.
• To engineer halogenase variants with improved activity and substrate scope for peptide and protein halogenation.

Main Methods:

• In vitro enzymatic halogenation (chlorination and bromination) of synthetic peptides.
• Identification of C-terminal peptide motifs recognized by halogenases.
• Rational design and engineering of PyrH halogenase mutants.
• Testing engineered halogenases on free peptides and genetically fused model proteins.

Main Results:

• Demonstrated novel in vitro halogenation of peptides, identifying the C-terminal (G/S)GW motif as a preferred substrate.
• Achieved chemo-catalyzed derivatization of an enzymatically chlorinated peptide.
• Engineered PyrH halogenase mutants exhibited improved halogenation of the (G/S)GW motif in peptides and proteins, reaching up to 90% efficiency.

Conclusions:

• Bacterial halogenases can be adapted for in vitro halogenation of peptides.
• Engineered halogenases, particularly PyrH variants, show significant potential for targeted peptide and protein halogenation.
• This work paves the way for advanced protein engineering and the development of novel halogenated biomolecules.