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

• Published in: Journal of molecular biology 2000-10, Vol.303 (1), p.77-91
• Main Authors: Rosano, Camillo, Bisso, Angela, Izzo, Gaetano, Tonetti, Michela, Sturla, Laura, De Flora, Antonio, Bolognesi, Martino
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution - genetics; Binding Sites; Carbohydrate Epimerases - chemistry; Carbohydrate Epimerases - genetics; Carbohydrate Epimerases - metabolism; Catalysis; Chromatography, Thin Layer; Crystallography, X-Ray; Deoxy Sugars - analysis; Deoxy Sugars - metabolism; Enzyme Stability; enzyme structure; epimerization; Escherichia coli - enzymology; Escherichia coli Proteins; Fucose - analogs & derivatives; Fucose - chemistry; Fucose - metabolism; GDP- l-fucose; Guanosine Diphosphate Mannose - analogs & derivatives; Guanosine Diphosphate Mannose - chemistry; Guanosine Diphosphate Mannose - metabolism; Holoenzymes - chemistry; Holoenzymes - genetics; Holoenzymes - metabolism; Hydrogen Bonding; Ketone Oxidoreductases; Kinetics; Models, Molecular; Multienzyme Complexes - chemistry; Multienzyme Complexes - genetics; Multienzyme Complexes - metabolism; Mutagenesis, Site-Directed - genetics; Mutation - genetics; NADP; NADP - metabolism; Protein Conformation; Protons; short-chain dehydrogenase; Structure-Activity Relationship; Substrate Specificity; Sugar Alcohol Dehydrogenases - chemistry; Sugar Alcohol Dehydrogenases - genetics; Sugar Alcohol Dehydrogenases - metabolism
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.2000.4106

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.