Laboratory Evolution of Metalloid Reductase Substrate Specificity and Product Size

13 June 2023, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


: 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.


cloneable nanoparticles

Supplementary materials

SI of Laboratory Evolution of Metalloid Substrate Recognition and Nanoparticle Product Size
Methods etc. for the manuscript submission titled 'Laboratory Evolution of Metalloid Substrate Recognition and Nanoparticle Product Size


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