title: Structure and function of GDP-mannose-3',5'-epimerase: an enzyme which performs three chemical reactions at the same active site.
abstract: GDP-mannose-3',5'-epimerase (GME) from Arabidopsis thaliana catalyzes the epimerization of both the 3' and 5' positions of GDP-alpha-D-mannose to yield GDP-beta-L-galactose. Production of the C5' epimer of GDP-alpha-D-mannose, GDP-beta-L-gulose, has also been reported. The reaction occurs as part of vitamin C biosynthesis in plants. We have determined structures of complexes of GME with GDP-alpha-D-mannose, GDP-beta-L-galactose, and a mixture of GDP-beta-L-gulose with GDP-beta-L-4-keto-gulose to resolutions varying from 2.0 to 1.4 A. The enzyme has the classical extended short-chain dehydratase/reductase (SDR) fold. We have confirmed that GME establishes an equilibrium between two products, GDP-beta-L-galactose and GDP-beta-L-gulose. The reaction proceeds by C4' oxidation of GDP-alpha-D-mannose followed by epimerization of the C5' position to give GDP-beta-L-4-keto-gulose. This intermediate is either reduced to give GDP-beta-L-gulose or the C3' position is epimerized to give GDP-beta-L-4-keto-galactose, then C4' is reduced to GDP-beta-L-galactose. The combination of oxidation, epimerization, and reduction in a single active site is unusual. Structural analysis coupled to site-directed mutagenesis suggests C145 and K217 as the acid/base pair responsible for both epimerizations. On the basis of the structure of the GDP-beta-L-gulose/GDP-beta-L-4-keto-gulose co-complex, we predict that a ring flip occurs during the first epimerization and that a boat intermediate is likely for the second epimerization. Comparison of GME with other SDR enzymes known to abstract a protein alpha to the keto function of a carbohydrate identifies key common features.
authors.affiliation: Centre for Biomolecular Sciences, University of St. Andrews, North Haugh, St. Andrews, Fife, Scotland KY16 9ST, United Kingdom.
authors.forename: Louise L
authors.initials: LL
authors.lastname: Major
authors.forename: Beata A
authors.initials: BA
authors.lastname: Wolucka
authors.forename: James H
authors.initials: JH
authors.lastname: Naismith
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology; D001665:Binding Sites; D002238:Carbohydrate Epimerases / Q000737:chemistry* / Q000378:metabolism*; D006155:Guanosine Diphosphate Mannose / Q000737:chemistry* / Q000378:metabolism*; D006156:Guanosine Diphosphate Sugars / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D013329:Structure-Activity Relationship
chemical_list: C480160:GDP-gulose; D006156:Guanosine Diphosphate Sugars; D009243:NAD; D006155:Guanosine Diphosphate Mannose; D002238:Carbohydrate Epimerases; C019192:GDPmannose 3,5-epimerase