title: Purification and properties of the Escherichia coli K-12 NAD-dependent nucleotide diphosphosugar epimerase, ADP-L-glycero-D-mannoheptose 6-epimerase.
abstract: The Escherichia coli K-12 NAD-dependent nucleotide-diphosphosugar epimerase, ADP-L-glycero-D-mannoheptose 6-epimerase, catalyzes the conversion of ADP-D-glycero-D-mannoheptose to ADP-L-glycero-D-mannoheptose. ADP-L-glycero-D-mannoheptose is a key intermediate of lipopolysaccharide inner core biosynthesis in several genera of Gram-negative bacteria. Sedimentation equilibrium and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified epimerase revealed that the native enzyme has a molecular mass of 240 kDa and a subunit molecular weight of 37,000 +/- 3,000. Lectin binding studies of the purified epimerase indicated that the protein is glycosylated. There was 1 mol of tightly bound NAD+ per enzyme subunit. Variable but small fractions of purified preparations of epimerase are highly fluorescent and contain NADH. The native enzyme can be resolved into apoenzyme and NAD+ by acidic ammonium sulfate precipitation. The catalytic activity can be reconstituted with the addition of NAD+ to the apoenzyme. Optimum pH range for enzyme activity is broad, between 5.5 and 9.5. It exhibits a temperature optimum at 42 degrees C. The Km and Vmax for the substrate is 0.1 mM and 46 mumol 30 min-1 mg-1, respectively. The native enzyme displays UV and fluorescence spectra that are consistent with the presence of enzyme bound NAD+. CD spectra of the holoepimerase indicate 11% alpha-helical and 36% beta-sheet structures.
authors.affiliation: Section on Pharmacology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892.
authors.forename: L
authors.initials: L
authors.lastname: Ding
authors.forename: B L
authors.initials: BL
authors.lastname: Seto
authors.forename: S A
authors.initials: SA
authors.lastname: Ahmed
authors.forename: W G
authors.initials: WG
authors.lastname: Coleman
mesh_terms: D000595:Amino Acid Sequence; D002238:Carbohydrate Epimerases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000032:analysis; D002384:Catalysis; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism*; D017433:Protein Structure, Secondary
chemical_list: D002241:Carbohydrates; D009243:NAD; C037213:ADP-L-glycero-D-mannoheptose-6-epimerase; D002238:Carbohydrate Epimerases