1e7q: Difference between revisions

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.

1E7Q is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with SO4, NAP, UVW and TRS as [http://en.wikipedia.org/wiki/ligands ligands]. Structure known Active Sites: AC1 and AC2. Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1E7Q OCA].  

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