Expanding the Nucleotide and Sugar 1-Phosphate Promiscuity of Nucleotidyltransferase RmlA via Directed Evolution
- UW
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 a- and b-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.
- Research Organization:
- Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
- Sponsoring Organization:
- National Institutes of Health (NIH)
- OSTI ID:
- 1031368
- Journal Information:
- J. Biol. Chem., Vol. 286, Issue (15) ; 04, 2011; ISSN 0021-9258
- Country of Publication:
- United States
- Language:
- ENGLISH
Similar Records
In vitro bypass of UV-induced lesions by Escherichia coli DNA polymerase I: specificity of nucleotide incorporation
UTP-Bound And Apo Structures of a Minimal RNA Uridylyltransferase