Directed evolution of APOX for proximity labeling using phenols with high redox potentials

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• Proximity labeling (PL) identifies proteins in specific subcellular regions using enzymes like APEX2 (enhanced ascorbate peroxidase 2).
• APEX2 oxidizes biotin phenol (BP) into a radical that tags nearby proteins, but its large diffusion radius limits spatial resolution.
• Existing PL enzymes struggle to oxidize high-redox-potential substrates like nitrophenol (NP), hindering shorter labeling radii.

Purpose of the Study:

• To engineer a novel proximity labeling enzyme with improved substrate oxidation capabilities and higher spatial resolution.
• To overcome the limitations of APEX2's radical diffusion radius for more precise subcellular proteomic mapping.

Main Methods:

• Ultrahigh-throughput directed evolution was employed to create APOX (APEX with high redOX potential), a quadruple mutant of APEX2.
• APOX exhibits a higher reduction potential, enabling efficient oxidation of high-redox-potential substrates.
• The study utilized a membrane-permeable alkyne-NP probe with APOX for proximity labeling in living mammalian cells.

Main Results:

• APOX demonstrated efficient proximity labeling in living mammalian cells.
• Proteomic mapping using APOX and the NP probe achieved excellent subcellular compartment specificity.
• The engineered enzyme successfully addressed the spatial resolution limitations of previous PL methods.

Conclusions:

• APOX represents a significant advancement in proximity labeling technology, offering enhanced spatial resolution.
• This new enzyme facilitates precise proteomic mapping within specific subcellular compartments in living cells.
• APOX enables more accurate identification of endogenous proteins with improved spatiotemporal control.