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

J Mol Biol. 2000 Oct 13;303(1):77-91. 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 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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
  • Escherichia coli / enzymology*
  • Escherichia coli Proteins*
  • Fucose / analogs & derivatives
  • Fucose / chemistry
  • Fucose / metabolism
  • 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 / metabolism
  • Protein Conformation
  • Protons
  • Structure-Activity Relationship
  • Substrate Specificity
  • Sugar Alcohol Dehydrogenases / chemistry*
  • Sugar Alcohol Dehydrogenases / genetics
  • Sugar Alcohol Dehydrogenases / metabolism*


  • Deoxy Sugars
  • Escherichia coli Proteins
  • Holoenzymes
  • Multienzyme Complexes
  • Protons
  • wcaG protein, E coli
  • GDP-4-keto-6-deoxymannose
  • Fucose
  • Guanosine Diphosphate Mannose
  • NADP
  • Sugar Alcohol Dehydrogenases
  • Ketone Oxidoreductases
  • Carbohydrate Epimerases

Associated data

  • PDB/1E6U
  • PDB/1E7Q
  • PDB/1E7R
  • PDB/1E7S