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1e7q
From Proteopedia
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'''GDP 4-KETO-6-DEOXY-D-MANNOSE EPIMERASE REDUCTASE S107A''' | '''GDP 4-KETO-6-DEOXY-D-MANNOSE EPIMERASE REDUCTASE S107A''' | ||
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[[Category: Izzo, G.]] | [[Category: Izzo, G.]] | ||
[[Category: Rosano, C.]] | [[Category: Rosano, C.]] | ||
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Revision as of 11:46, 2 May 2008
GDP 4-KETO-6-DEOXY-D-MANNOSE EPIMERASE REDUCTASE S107A
Overview
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.
About this Structure
1E7Q is a Single protein structure of sequence from Escherichia coli. Full crystallographic information is available from OCA.
Reference
Probing the catalytic mechanism of GDP-4-keto-6-deoxy-d-mannose Epimerase/Reductase by kinetic and crystallographic characterization of site-specific mutants., Rosano C, Bisso A, Izzo G, Tonetti M, Sturla L, De Flora A, Bolognesi M, J Mol Biol. 2000 Oct 13;303(1):77-91. PMID:11021971 Page seeded by OCA on Fri May 2 14:46:07 2008
