1E7Q

GDP 4-keto-6-deoxy-D-mannose epimerase reductase S107A

Summary for 1E7Q

• Entry DOI: 10.2210/pdb1e7q/pdb
• Related: 1BSV 1BWS 1E6U 1E7R 1E7S 1FXS 1GFS
• Descriptor: GDP-FUCOSE SYNTHETASE, NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE, ACETYLPHOSPHATE, ... (6 entities in total)
• Functional Keywords: epimerase/reductase, sdr, red, epimerase-reductase complex
• Biological source: ESCHERICHIA COLI
• Total number of polymer chains: 1
• Total formula weight: 37517.91

Authors

Rosano, C.,Izzo, G.,Bolognesi, M. (deposition date: 2000-09-07, release date: 2000-10-18, Last modification date: 2023-12-13)

Primary citation

Rosano, C.,Bisso, A.,Izzo, G.,Tonetti, M.,Sturla, L.,De Flora, A.,Bolognesi, M.
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-, 2000

PubMed 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.

PubMed: 11021971
DOI: 10.1006/JMBI.2000.4106

Experimental method

X-RAY DIFFRACTION (1.6 Å)