Laboratory Evolution of Metalloid Reductase Substrate Recognition and Nanoparticle Product Size

Area of Science:

• Biocatalysis and Enzyme Engineering
• Nanomaterial Biosynthesis
• Metalloid Metabolism

Background:

• Glutathione reductase-like metalloid reductase (GRLMR) naturally reduces selenodiglutathione to form selenium nanoparticles (SeNPs).
• Directed evolution offers a powerful approach to engineer enzyme properties for novel applications.
• Understanding enzyme active site dynamics is crucial for protein engineering.

Purpose of the Study:

• To engineer GRLMR variants with improved catalytic activity for selenodiglutathione reduction.
• To explore the potential of directed evolution for creating novel metalloid-processing enzymes.
• To investigate the substrate specificity and catalytic capabilities of evolved GRLMR variants.

Main Methods:

• Generated a large library of GRLMR variants using error-prone PCR.
• Expressed and screened variants in *Escherichia coli* under selenite (SeO3^2-) challenge.
• Characterized the enzymatic activity of the selected variant using various substrates and NADPH.

Main Results:

• Isolated a GRLMR variant (named SeR) with two mutations, one near the active site dicysteine.
• The evolved SeR exhibited enhanced reduction of selenite (SeO3^2-) and gained the ability to reduce selenate (SeO4^2-).
• SeR demonstrated broader substrate specificity compared to the parent GRLMR.

Conclusions:

• Laboratory-directed evolution can successfully engineer enzymes for enhanced metalloid reduction and novel substrate utilization.
• The evolved selenium reductase (SeR) provides a new biocatalyst for selenium nanoparticle biosynthesis.
• This study highlights the potential for creating new biosynthetic routes for inorganic nanomaterials.