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.