Engineered Biosynthesis of β-Alkyl Tryptophan Analogues

Area of Science:

• Biocatalysis and Enzyme Engineering
• Synthetic Organic Chemistry
• Medicinal Chemistry

Background:

• Noncanonical amino acids (ncAAs) with α and β stereocenters are crucial for synthesizing natural products and therapeutics.
• Current methods for preparing these β-branched ncAAs are often inefficient and multistep, limiting their accessibility.
• There is a need for streamlined, stereoselective synthetic routes to these valuable compounds.

Purpose of the Study:

• To develop a novel biocatalytic method for the stereoselective synthesis of β-branched noncanonical amino acids.
• To engineer a variant of Pyrococcus furiosus tryptophan synthase (PfTrpB) for enhanced catalytic activity and substrate scope.
• To establish an efficient and environmentally benign platform for producing β-branched tryptophan analogues.

Main Methods:

• Enzyme engineering of Pyrococcus furiosus tryptophan synthase (PfTrpB) to create the variant PfTrpB7E6.
• Stereoselective biocatalytic synthesis of β-branched tryptophan analogues using PfTrpB7E6.
• Characterization of PfTrpB7E6 through X-ray crystallography and UV/Vis spectroscopy to elucidate the molecular basis of its activity.
• Synthesis and characterization of 27 distinct noncanonical amino acids.

Main Results:

• The engineered enzyme PfTrpB7E6 successfully synthesized bulky β-branched tryptophan analogues in a single step.
• PfTrpB7E6 demonstrated broad substrate tolerance, providing access to 27 different noncanonical amino acids.
• Structural and spectroscopic analyses revealed that specific mutations enhance the abundance and persistence of a key reactive intermediate, leading to efficient catalysis.
• The biocatalytic process is operationally simple and environmentally benign.

Conclusions:

• PfTrpB7E6 represents the first biocatalyst capable of synthesizing bulky β-branched tryptophan analogues in one step.
• This engineered enzyme provides a versatile and sustainable platform for the preparation of diverse β-branched tryptophan building blocks.
• The findings offer a significant advancement in the synthesis of noncanonical amino acids for applications in natural product synthesis and drug discovery.