PDBsum entry 1e7r

Isomerase

PDB id: 1e7r

Contents

Protein chain

314 a.a. *

Ligands

NAP

UVW

TRS ×2

SO4 ×2

Waters ×347

* Residue conservation analysis

PDB id:

1e7r

Name:

Isomerase

Title:

Gdp 4-keto-6-deoxy-d-mannose epimerase reductase y136e

Structure:

Gdp-fucose synthetase. Chain: a. Synonym: gdp-4-keto 6-deoxy-mannose 3,5-epimerase 4-reductase, gmer. Engineered: yes. Mutation: yes. Other_details: unknown molecule labeled as acetylphosphate

Source:

Escherichia coli. Organism_taxid: 562. Expressed in: escherichia coli. Expression_system_taxid: 562

Biol. unit:

Dimer (from PDB file)

Resolution:

1.60Å R-factor: 0.139 R-free: 0.180

Authors:

C.Rosano,G.Izzo,M.Bolognesi

Key ref:

C.Rosano et al. (2000). Probing the catalytic mechanism of GDP-4-keto-6-deoxy-d-mannose Epimerase/Reductase by kinetic and crystallographic characterization of site-specific mutants. J Mol Biol, 303, 77-91. PubMed id: 11021971 DOI: 10.1006/jmbi.2000.4106

Date:

07-Sep-00 Release date: 18-Oct-00

Protein chain

P32055  (FCL_ECOLI) -  GDP-L-fucose synthase from Escherichia coli (strain K12)

Seq: 321 a.a.

Struc: 314 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.1.1.1.271 - GDP-L-fucose synthase.
• Pathway: GDP-L-Fucose and GDP-mannose Biosynthesis
• Reaction: GDP-beta-L-fucose + NADP⁺ = GDP-4-dehydro-alpha-D-rhamnose + NADPH + H⁺

GDP-beta-L-fucose + NADP(+) (Bound ligand (Het Group name = NAP) corresponds exactly) = GDP-4-dehydro-alpha-D-rhamnose + NADPH + H(+)

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

reference

DOI no: 10.1006/jmbi.2000.4106

J Mol Biol 303:77-91 (2000)

PubMed id: 11021971

Probing the catalytic mechanism of GDP-4-keto-6-deoxy-d-mannose Epimerase/Reductase by kinetic and crystallographic characterization of site-specific mutants.

C.Rosano, A.Bisso, G.Izzo, M.Tonetti, L.Sturla, A.De Flora, M.Bolognesi.

ABSTRACT

GDP-4-keto-6-deoxy-d-mannose epimerase/reductase is a bifunctional enzyme responsible for the last step in the biosynthesis of GDP-l-fucose, the substrate of fucosyl transferases. Several cell-surface antigens, including the leukocyte Lewis system and cell-surface antigens in pathogenic bacteria, depend on the availability of GDP-l-fucose for their expression. Therefore, the enzyme is a potential target for therapy in pathological states depending on selectin-mediated cell-to-cell interactions. Previous crystallographic investigations have shown that GDP-4-keto-6-deoxy-d-mannose epimerase/reductase belongs to the short-chain dehydrogenase/reductase protein homology family. The enzyme active-site region is at the interface of an N-terminal NADPH-binding domain and a C-terminal domain, held to bind the substrate. The design, expression and functional characterization of seven site-specific mutant forms of GDP-4-keto-6-deoxy-d-mannose epimerase/reductase are reported here. In parallel, the crystal structures of the native holoenzyme and of three mutants (Ser107Ala, Tyr136Glu and Lys140Arg) have been investigated and refined at 1. 45-1.60 A resolution, based on synchrotron data (R-factors range between 12.6 % and 13.9 %). The refined protein models show that besides the active-site residues Ser107, Tyr136 and Lys140, whose mutations impair the overall enzymatic activity and may affect the coenzyme binding mode, side-chains capable of proton exchange, located around the expected substrate (GDP-4-keto-6-deoxy-d-mannose) binding pocket, are selectively required during the epimerization and reduction steps. Among these, Cys109 and His179 may play a primary role in proton exchange between the enzyme and the epimerization catalytic intermediates. Finally, the additional role of mutated active-site residues involved in substrate recognition and in enzyme stability has been analyzed.

Selected figure(s)

Figure 1.
Figure 1. The GDP- Image -fucose biosynthetic pathway and the products obtained after NaBH[4] reduction of the intermediate compounds. GMD, GDP- Image -mannose 4,6 dehydratase; GMER, GDP-4-keto-6-deoxy- Image -mannose epimerase/reductase; R-, GDP.

Figure 6.
Figure 6. Stereo view of the proposed binding mode for a nucleotide-sugar molecule (shown as a space-filling model) relative to active-site residues discussed in the text. The C-2 and C-3 centers can be recognized as those closest to His179 side-chain; the C-4 center falls next to the nicotinamide carboxamido group.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2000, 303, 77-91) copyright 2000.

Figures were selected by an automated process.