PDBsum entry 1mp4

Transferase

PDB id: 1mp4

Contents

Protein chains

289 a.a. *

Ligands

UPG ×2

* Residue conservation analysis

PDB id:

1mp4

Name:

Transferase

Title:

W224h variant of s. Enterica rmla

Structure:

W224h variant of s. Enterica rmla bound to udp-glucose. Chain: a, b. Synonym: thymidylyltransferase. Engineered: yes. Mutation: yes

Source:

Salmonella enterica. Organism_taxid: 28901. Expressed in: escherichia coli. Expression_system_taxid: 562

Biol. unit:

Tetramer (from PDB file)

Resolution:

2.20Å R-factor: 0.257 R-free: 0.288

Authors:

W.A.Barton,J.B.Biggins,J.Jiang,J.S.Thorson,D.B.Nikolov

Key ref:

W.A.Barton et al. (2002). Expanding pyrimidine diphosphosugar libraries via structure-based nucleotidylyltransferase engineering. Proc Natl Acad Sci U S A, 99, 13397-13402. PubMed id: 12374866 DOI: 10.1073/pnas.192468299

Date:

11-Sep-02 Release date: 09-Oct-02

Protein chains

Q9F7G8  (Q9F7G8_SALER) -  Glucose-1-phosphate thymidylyltransferase from Salmonella enterica

Seq: 292 a.a.

Struc: 289 a.a.*

* PDB and UniProt seqs differ at 16 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.2.7.7.24 - glucose-1-phosphate thymidylyltransferase.
• Pathway: 6-Deoxyhexose Biosynthesis
• Reaction: dTTP + alpha-D-glucose 1-phosphate + H⁺ = dTDP-alpha-D-glucose + diphosphate

dTTP + alpha-D-glucose 1-phosphate + H(+) = dTDP-alpha-D-glucose + diphosphate (Bound ligand (Het Group name = UPG) matches with 94.59% similarity)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1073/pnas.192468299

Proc Natl Acad Sci U S A 99:13397-13402 (2002)

PubMed id: 12374866

Expanding pyrimidine diphosphosugar libraries via structure-based nucleotidylyltransferase engineering.

W.A.Barton, J.B.Biggins, J.Jiang, J.S.Thorson, D.B.Nikolov.

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.

Selected figure(s)

Figure 1.
Fig 1. (a) The reaction catalyzed by E[p]. (b) Sugar phosphates used for this study. The corresponding deviations from the natural substrate (2) are highlighted in red.

Figure 2.
Fig 2. Schematic representation of E[p] and glucose active-site interactions using the program LIGPLOT (19). The glucose moiety of the product, UDP-glucose, and the residues that interact with it in the E[p] active site are shown in ball-and-stick format. Hydrogen bonds are illustrated as dashed lines, and hydrophobic interactions are indicated by half circles.

Figures were selected by an automated process.