Biocatalytic Bamberger Rearrangement for the Synthesis of 3-Amino-2-hydroxyacetophenone via Hydroxylaminobenzene...

Area of Science:

• Biocatalysis and Enzyme Engineering
• Metabolic Engineering
• Pharmaceutical Synthesis

Background:

• The Bamberger rearrangement is crucial for synthesizing functionalized aminophenols used in pharmaceuticals.
• Hydroxylaminobenzene mutase (HabM) performs a key isomerization for Pranlukast intermediate biosynthesis but suffers from low efficiency and an unknown structure.
• Rational engineering of HabM and its associated pathways is essential for improving the production of valuable biopharmaceutical intermediates.

Purpose of the Study:

• To elucidate the structure and function of Hydroxylaminobenzene mutase (HabM).
• To engineer an efficient multi-enzyme cascade for the biosynthesis of 3-amino-2-hydroxyacetophenone (3AHAP).
• To establish a paradigm for optimizing complex enzymatic pathways for sustainable biopharmaceutical production.

Main Methods:

• AI-assisted phylogenetic mining and deep learning-guided screening to identify optimal enzyme combinations (NR^Bh-HabM^Eo).
• Integrated structural elucidation (spectroscopy, SEC, AlphaFold3) and protein engineering (mutagenesis, molecular dynamics) of HabM^Eo.
• Metabolic engineering strategies including NAD kinase introduction and RIAD/RIDD-based scaffolding for pathway optimization and intermediate channeling.

Main Results:

• Identified and characterized HabM^Eo as a Fe-dependent tetramer, revealing its structural basis.
• Engineered a synergistic triple mutant of HabM with significantly enhanced catalytic efficiency.
• Developed a spatially organized multi-enzyme cascade using scaffolding, improving intermediate channeling and suppressing over-reduction, leading to enhanced 3AHAP biosynthesis.

Conclusions:

• The study successfully elucidated the structure of HabM and its mechanism.
• Protein engineering and metabolic coordination strategies led to a highly efficient multi-enzyme cascade for 3AHAP production.
• This work provides a successful paradigm for optimizing complex enzymatic cascades for the sustainable manufacturing of high-value biopharmaceutical intermediates.