TY - JOUR
T1 - Probing the catalytic mechanism of GDP-4-keto-6-deoxy-D-mannose epimerase/reductase by kinetic and crystallographic characterization of site-specific mutants
AU - Rosano, Camillo
AU - Bisso, Angela
AU - Izzo, Gaetano
AU - Tonetti, Michela
AU - Sturla, Laura
AU - De Flora, Antonio
AU - Bolognesi, Martino
PY - 2000/10/13
Y1 - 2000/10/13
N2 - 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 Å 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. (C) 2000 Academic Press.
AB - 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 Å 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. (C) 2000 Academic Press.
KW - Enzyme structure
KW - Epimerization
KW - GDP-L-fucose
KW - NADP
KW - Short-chain dehydrogenase
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U2 - 10.1006/jmbi.2000.4106
DO - 10.1006/jmbi.2000.4106
M3 - Article
C2 - 11021971
AN - SCOPUS:0034644773
VL - 303
SP - 77
EP - 91
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 1
ER -