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Using Domain Insertion to Create Sulfite Reductases That Present Chemical-Dependent Activities
Elizabeth Windham1, Dru Myerscough1, Samuel K Schwartz2
1Department of Biosciences, Rice University, 6100 Main Street, Houston, Texas 77005, United States.
ACS Synthetic Biology
|May 20, 2026
Summary
Protein engineering using domain insertion can create analyte-dependent oxidoreductases. This study engineered large sulfite reductases (SiR) for analyte sensing and environmental monitoring.
Area of Science:
- Protein Engineering
- Biocatalysis
- Enzyme Activity
Background:
- Domain insertion is a protein engineering strategy to create analyte-dependent oxidoreductases.
- Applying domain insertion to large, hetero-oligomeric proteins with dynamic conformational changes remains challenging.
Purpose of the Study:
- To investigate the effects of peptide and domain insertions on the activity of NADPH-dependent sulfite reductase (SiR) from Escherichia coli.
- To determine how to apply domain insertion to large hetero-oligomeric proteins.
Main Methods:
- Systematic octapeptide insertion and cellular selection to evaluate SiR mutational tolerance.
- Insertion of a ligand-binding domain at peptide-insertion-tolerant sites in SiR.
- Utilizing a bioelectrochemical reactor to monitor sulfide production.
Main Results:
- Identified regions in SiR tolerant to octapeptide insertion, retaining parent-like activity.
- Approximately 90% of domain insertion variants retained catalytic activity.
- >50% of variants showed activity regulated by an endocrine disruptor.
- Demonstrated conditional sulfide production monitored electrochemically.
Conclusions:
- Systematic peptide insertion can guide domain insertion in large protein complexes like SiR.
- Engineered SiR can convert environmental chemical information into a redox-active metabolite.
- This approach enables environmental sensing and monitoring through bioelectrochemical systems.
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