In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 99, No. 21 ( 2002-10-15), p. 13397-13402
Abstract:
In vitro “glycorandomization” is a chemoenzymatic approach for generating diverse libraries of glycosylated biomolecules based on natural product scaffolds. This technology makes use of engineered variants of specific enzymes affecting metabolite glycosylation, particularly nucleotidylyltransferases and glycosyltransferases. To expand the repertoire of UDP/dTDP sugars readily available for glycorandomization, we now report a structure-based engineering approach to increase the diversity of α- d -hexopyranosyl phosphates accepted by Salmonella enterica LT2 α- d -glucopyranosyl phosphate thymidylyltransferase (E p ). This article highlights the design rationale, determined substrate specificity, and structural elucidation of three “designed” mutations, illustrating both the success and unexpected outcomes from this type of approach. In addition, a single amino acid substitution in the substrate-binding pocket (L89T) was found to significantly increase the set of α- d -hexopyranosyl phosphates accepted by E p to include α- d - allo -, α- d - altro -, and α- d - talo pyranosyl phosphate. In aggregate, our results provide valuable blueprints for altering nucleotidylyltransferase specificity by design, which is the first step toward in vitro glycorandomization.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.192468299
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2002
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
