Laboratory Evolution of Metalloid Reductase Substrate Recognition and Nanoparticle Product Size.

Glutathione reductase-like metalloid reductase (GRLMR) is an enzyme that reduces selenodiglutathione (GS-Se-SG), forming zerovalent Se nanoparticles (SeNPs). Error-prone polymerase chain reaction was used to create a library of ∼10,000 GRLMR variants. The library was expressed in BL21 Escherichia coli in liquid culture with 50 mM of SeO 3 2- present, under the hypothesis that the enzyme variants with improved GS-Se-SG reduction kinetics would emerge. The selection resulted in a GRLMR variant with two mutations. One of the mutations (D-E) lacks an obvious functional role, whereas the other mutation is L-H within 5 Å of the enzyme active site. This mutation places a second H residue within 5 Å of an active site dicysteine. This GRLMR variant was characterized for NADPH-dependent reduction of GS-Se-SG, GSSG, SeO 3 2- , SeO 4 2- , GS-Te-SG, and TeO 3 2- . The evolved enzyme demonstrated enhanced reduction of SeO 3 2- and gained the ability to reduce SeO 4 2- . This variant is named selenium reductase (SeR) because of its emergent broad activity for a wide variety of Se substrates, whereas the parent enzyme was specific for GS-Se-SG. This study overall suggests that new biosynthetic routes are possible for inorganic nanomaterials using laboratory-directed evolution methods.