Trapping biosynthetic acyl-enzyme intermediates with encoded 2,3-diaminopropionic acid

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Enzymes frequently catalyze reactions via transient acyl-enzyme intermediates (esters or thioesters).
• These intermediates are often unstable, with half-lives ranging from minutes to hours.
• Existing methods for stabilizing these complexes often yield non-native structures.

Purpose of the Study:

• To develop a novel strategy for stabilizing acyl-enzyme intermediates.
• To enable the capture and structural analysis of transiently formed enzyme-substrate complexes.
• To investigate the catalytic mechanisms of enzymes involved in peptide synthesis.

Main Methods:

• Incorporation of 2,3-diaminopropionic acid (DAP) into recombinant proteins via genetic code expansion.
• Replacement of catalytic serine or cysteine residues with DAP to form stable amide bonds with substrates.
• Structural elucidation of enzyme intermediates using the DAP-based capture strategy.

Main Results:

• Successfully generated stable acyl-enzyme complexes by replacing active site residues with DAP.
• Captured and characterized the first and last acyl-thioesterase intermediates in valinomycin biosynthesis.
• Provided structural insights into the conformational changes governing substrate processing in nonribosomal peptide synthetases.

Conclusions:

• The DAP incorporation strategy provides a powerful tool for capturing and characterizing unstable acyl-enzyme intermediates.
• This method facilitates the structural and mechanistic study of enzymes, including those in complex biosynthetic pathways.
• The approach holds promise for identifying the native substrates of transiently acylated proteins with unknown functions.