2-Ketogluconate Kinase from Cupriavidus necator H16: Purification, Characterization, and Exploration of Its Substrate Specificity

We have cloned, overexpressed, purified, and characterized a 2-ketogluconate kinase (2-dehydrogluconokinase, EC 2.7.1.13) from Cupriavidus necator (Ralstonia eutropha) H16. Exploration of its substrate specificity revealed that three ketoacids (2-keto-3-deoxy-d-gluconate, 2-keto-d-gulonate, and 2-keto-3-deoxy-d-gulonate) with structures close to the natural substrate (2-keto-d-gluconate) were successfully phosphorylated at an efficiency lower than or comparable to 2-ketogluconate, as depicted by the measured kinetic constant values. Eleven aldo and keto monosaccharides of different chain lengths and stereochemistries were also assayed but not found to be substrates. 2-ketogluconate-6-phosphate was synthesized at a preparative scale and was fully characterized for the first time.