title: Expanding the nucleotide and sugar 1-phosphate promiscuity of nucleotidyltransferase RmlA via directed evolution.
abstract: Directed evolution is a valuable technique to improve enzyme activity in the absence of a priori structural knowledge, which can be typically enhanced via structure-guided strategies. In this study, a combination of both whole-gene error-prone polymerase chain reaction and site-saturation mutagenesis enabled the rapid identification of mutations that improved RmlA activity toward non-native substrates. These mutations have been shown to improve activities over 10-fold for several targeted substrates, including non-native pyrimidine- and purine-based NTPs as well as non-native D- and L-sugars (both α- and β-isomers). This study highlights the first broadly applicable high throughput sugar-1-phosphate nucleotidyltransferase screen and the first proof of concept for the directed evolution of this enzyme class toward the identification of uniquely permissive RmlA variants.
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
authors.forename: Rocco
authors.initials: R
authors.lastname: Moretti
authors.forename: Aram
authors.initials: A
authors.lastname: Chang
authors.forename: Pauline
authors.initials: P
authors.lastname: Peltier-Pain
authors.forename: Craig A
authors.initials: CA
authors.lastname: Bingman
authors.forename: George N
authors.initials: GN
authors.lastname: Phillips
authors.forename: Jon S
authors.initials: JS
authors.lastname: Thorson
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D019020:Directed Molecular Evolution; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019779:Salmonella enterica / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D009713:Nucleotidyltransferases; C020587:glucose-1-phosphate thymidylyltransferase