1MP4: W224H VARIANT OF S. ENTERICA RmlA

Citation:
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 alpha-d-hexopyranosyl phosphates accepted by Salmonella enterica LT2 alpha-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 alpha-d-hexopyranosyl phosphates accepted by E(p) to include alpha-d-allo-, alpha-d-altro-, and alpha-d-talopyranosyl phosphate. In aggregate, our results provide valuable blueprints for altering nucleotidylyltransferase specificity by design, which is the first step toward in vitro glycorandomization.
PDB ID: 1MP4Download
MMDB ID: 20999
PDB Deposition Date: 2002/9/11
Updated in MMDB: 2012/12
Experimental Method:
x-ray diffraction
Resolution: 2.2  Å
Source Organism:
Similar Structures:
Biological Unit for 1MP4: tetrameric; determined by author
Molecular Components in 1MP4
Label Count Molecule
Proteins (4 molecules)
4
W224h Variant of S. Enterica Rmla Bound to Udp-glucose
Molecule annotation
Chemicals (4 molecules)
1
4
* Click molecule labels to explore molecular sequence information.

Citing MMDB
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