A colorimetric assay for the screening and kinetic analysis of nucleotide sugar 4,6-dehydratases

Nucleotide sugar 4,6-dehydratases belong to the Short-chain Dehydrogenase/Reductase (SDR) superfamily and catalyze the conversion of an NDP-hexose to an NDP-4-keto-6-deoxy hexose, a key step in the biosynthesis of a plethora of deoxy and amino sugars. Here, we present a colorimetric assay for the detection of their reaction products (NDP-4-keto-6-deoxy hexoses) using concentrated sulfuric acid and an ethanolic resorcinol solution. Under these conditions, the keto-function of the dehydratase product reacts specifically with resorcinol to form an orange-red or pink complex for NDP-glucose/GDP-mannose and UDP-N-acetylglucosamine, respectively, with an absorption maximum at 510 nm. The presented assay allows reliable product detection at low concentrations and can be applied in microtiter plates. It thus allows the determination of kinetic enzyme parameters like the optimal temperature, pH, Vmax, KM and kcat, as well as the miniaturization for screening purposes with crude cell extracts. As such, this detection assay opens new possibilities for the characterization and screening of these dehydratases in 96-well plates for different research goals.