title: Probing the catalytic mechanism of GDP-4-keto-6-deoxy-d-mannose Epimerase/Reductase by kinetic and crystallographic characterization of site-specific mutants.
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.
authors.affiliation: Department of Physics-INFM and Advanced Biotechnology Center-IST, University of Genova, Largo Rosanna Benzi 10, Genova, I-16132, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Rosano
authors.forename: A
authors.initials: A
authors.lastname: Bisso
authors.forename: G
authors.initials: G
authors.lastname: Izzo
authors.forename: M
authors.initials: M
authors.lastname: Tonetti
authors.forename: L
authors.initials: L
authors.lastname: Sturla
authors.forename: A
authors.initials: A
authors.lastname: De Flora
authors.forename: M
authors.initials: M
authors.lastname: Bolognesi
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites; D002238:Carbohydrate Epimerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D002855:Chromatography, Thin Layer; D018360:Crystallography, X-Ray; D003837:Deoxy Sugars / Q000032:analysis / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005643:Fucose / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D006155:Guanosine Diphosphate Mannose / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D020035:Holoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006860:Hydrogen Bonding; D007658:Ketone Oxidoreductases; D007700:Kinetics; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D009249:NADP / Q000378:metabolism; D011487:Protein Conformation; D011522:Protons; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013401:Sugar Alcohol Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D003837:Deoxy Sugars; D029968:Escherichia coli Proteins; D020035:Holoenzymes; D009097:Multienzyme Complexes; D011522:Protons; C110682:wcaG protein, E coli; C054686:GDP-4-keto-6-deoxymannose; D005643:Fucose; D006155:Guanosine Diphosphate Mannose; D009249:NADP; D013401:Sugar Alcohol Dehydrogenases; D007658:Ketone Oxidoreductases; D002238:Carbohydrate Epimerases