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Functional Complementation Analysis (FCA): A Laboratory Exercise Designed and Implemented to Supplement the Teaching of Biochemical Pathways
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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
PubMed
Summary
This summary is machine-generated.

Protein engineering using domain insertion can create analyte-dependent oxidoreductases. This study engineered large sulfite reductases (SiR) for analyte sensing and environmental monitoring.

Keywords:
bioelectronicsdomain insertionextracellular electron transferoxidoreductaseprotein engineeringsensorsulfite reductasesynthetic biology

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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.