Developing High Performance N-Oxygenase for Azomycin Synthesis through Ancestral Sequence Reconstruction

Area of Science:

• Biocatalysis and Enzyme Engineering
• Synthetic Biology
• Green Chemistry

Background:

• Traditional azomycin synthesis involves harsh conditions and low yields.
• Biocatalytic routes using N-oxygenase are limited by enzyme efficiency and stability.

Purpose of the Study:

• To overcome limitations in biocatalytic N-oxygenase synthesis.
• To develop a high-performance N-oxygenase for azomycin production using ancestral sequence reconstruction.

Main Methods:

• Resurrected and screened seven ancestral node proteins from 83 homologous sequences.
• Utilized ancestral sequence reconstruction (ASR) to identify improved enzyme variants.
• Performed molecular dynamics simulations to understand enhanced catalytic mechanisms.

Main Results:

• Engineered enzyme ASR-90 demonstrated a 546-fold increase in catalytic efficiency (kcat/Km) for 2-aminoimidazole.
• ASR-90 showed improved acid tolerance (optimal pH 4.5) and a 4-fold longer half-life at 50 °C.
• Achieved 50.9% substrate conversion in 20 min and displayed catalytic ability on 16 non-natural substrates.

Conclusions:

• Ancestral sequence reconstruction is an effective strategy for developing robust N-oxygenases.
• ASR-90 facilitates the green biosynthesis of azomycin and its derivatives.
• Enhanced substrate binding and active site modifications contribute to ASR-90's superior performance.