Laboratory Evolution of Metalloid Reductase Substrate Specificity and Product Size

Abstract

: 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 SeO32- 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 2 mutations. One of the mutations (D to E) lacks an obvious functional role, whereas the other mutation is L to H within 5 Å of the enzyme active site. This mutation places a second H residue within 5Å of an active site dicysteine. This GRLMR variant was characterized for NADPH dependent reduction of GS-Se-SG, GSSG, SeO32-, SeO42-, GS-Te-SG and TeO32-. The evolved enzyme demonstrated enhanced reduction of SeO32- and gained ability to reduce SeO42-. 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.