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  3. Developing High Performance N-oxygenase For Azomycin Synthesis Through Ancestral Sequence Reconstruction.
  1. Home
  3. Developing High Performance N-oxygenase For Azomycin Synthesis Through Ancestral Sequence Reconstruction.

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

Jinming Dou1, Bo Qin2, Zongjiang Yu3

  • 1State Key Laboratory Base of Eco-Chemical Engineering, College of Chemical Engineering, Qingdao University of Science and Technology, Qingdao 266101, China.

ACS Synthetic Biology
|June 3, 2026

View abstract on PubMed

Summary
This summary is machine-generated.

Ancestral sequence reconstruction engineered a superior N-oxygenase enzyme, ASR-90, for green azomycin synthesis. This biocatalyst shows enhanced efficiency, stability, and a broad substrate scope, overcoming limitations of traditional methods.

Keywords:
Ancestral sequence reconstructionAzomycinBiocatalysisN-OxygenaseNitro compounds

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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.