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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 ?- and ?-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.

SUBMITTER: Moretti R 

PROVIDER: S-EPMC3075670 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Expanding the nucleotide and sugar 1-phosphate promiscuity of nucleotidyltransferase RmlA via directed evolution.

Moretti Rocco R   Chang Aram A   Peltier-Pain Pauline P   Bingman Craig A CA   Phillips George N GN   Thorson Jon S JS  

The Journal of biological chemistry 20110211 15


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 substr  ...[more]

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