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Purification and properties of the Escherichia coli K-12 NAD-dependent nucleotide diphosphosugar epimerase, ADP-L-glycero-D-mannoheptose 6-epimerase. 1994

L Ding, and B L Seto, and S A Ahmed, and W G Coleman
Section on Pharmacology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892.

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.

UI MeSH Term Description Entries
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D009243 NAD A coenzyme composed of ribosylnicotinamide 5'-diphosphate coupled to adenosine 5'-phosphate by pyrophosphate linkage. It is found widely in nature and is involved in numerous enzymatic reactions in which it serves as an electron carrier by being alternately oxidized (NAD+) and reduced (NADH). (Dorland, 27th ed) Coenzyme I,DPN,Diphosphopyridine Nucleotide,Nadide,Nicotinamide-Adenine Dinucleotide,Dihydronicotinamide Adenine Dinucleotide,NADH,Adenine Dinucleotide, Dihydronicotinamide,Dinucleotide, Dihydronicotinamide Adenine,Dinucleotide, Nicotinamide-Adenine,Nicotinamide Adenine Dinucleotide,Nucleotide, Diphosphopyridine
D002238 Carbohydrate Epimerases Enzymes that catalyze the epimerization of chiral centers within carbohydrates or their derivatives. EC 5.1.3. Carbohydrate Isomerases,Epimerases, Carbohydrate,Isomerases, Carbohydrate
D002240 Carbohydrate Sequence The sequence of carbohydrates within POLYSACCHARIDES; GLYCOPROTEINS; and GLYCOLIPIDS. Carbohydrate Sequences,Sequence, Carbohydrate,Sequences, Carbohydrate
D002241 Carbohydrates A class of organic compounds composed of carbon, hydrogen, and oxygen in a ratio of Cn(H2O)n. The largest class of organic compounds, including STARCH; GLYCOGEN; CELLULOSE; POLYSACCHARIDES; and simple MONOSACCHARIDES. Carbohydrate
D002384 Catalysis The facilitation of a chemical reaction by material (catalyst) that is not consumed by the reaction. Catalyses
D002846 Chromatography, Affinity A chromatographic technique that utilizes the ability of biological molecules, often ANTIBODIES, to bind to certain ligands specifically and reversibly. It is used in protein biochemistry. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed) Chromatography, Bioaffinity,Immunochromatography,Affinity Chromatography,Bioaffinity Chromatography
D002852 Chromatography, Ion Exchange Separation technique in which the stationary phase consists of ion exchange resins. The resins contain loosely held small ions that easily exchange places with other small ions of like charge present in solutions washed over the resins. Chromatography, Ion-Exchange,Ion-Exchange Chromatography,Chromatographies, Ion Exchange,Chromatographies, Ion-Exchange,Ion Exchange Chromatographies,Ion Exchange Chromatography,Ion-Exchange Chromatographies
D002942 Circular Dichroism A change from planar to elliptic polarization when an initially plane-polarized light wave traverses an optically active medium. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed) Circular Dichroism, Vibrational,Dichroism, Circular,Vibrational Circular Dichroism
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli

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