Discovery of a Kojibiose Phosphorylase in Escherichia coli K-12

摘要

The substrate profiles for three uncharacterized enzymes (YcjM, YcjT, and YcjU) that are expressed from a cluster of twelve genes (ycjM-W and ompG) of unknown function in Escherichia coli K-12 have been determined. Through a comprehensive bioinformatic and steady-state kinetic analysis, the catalytic function of YcjT was determined to be kojibiose phosphorylase. In the presence of saturating phosphate and kojibiose (α-(1,2)-D-glucose-D-glucose) this enzyme catalyzes the formation of D-glucose and β-D-glucose-1-phosphate (kcat = 1.1 s⁻¹, Km = 1.05 mM, and kcat/Km = 1.12 x 10³ M⁻¹ s⁻¹). Additionally, it was also shown that in the presence of β-D-glucose-1-phosphate, YcjT can catalyze the formation of other disaccharides using 1,5-anhydro-D-glucitol, L-sorbose, D-sorbitol or L-iditol as a substitute for D-glucose. Kojibiose is a component of cell wall lipoteichoic acids in Gram-positive bacteria and is of interest as a potential low-calorie sweetener and prebiotic. YcjU was determined to be a β-phosphoglucomutase that catalyzes the isomerization of β-D-glucose-1-phosphate (kcat = 21 s⁻¹, Km = 18 μM, and kcat/Km = 1.1 x 10⁶ M⁻¹ s⁻¹) to D-glucose-6-phosphate. YcjU was also shown to exhibit catalytic activity with β-D-allose-1-phosphate, β-D-mannose-1-phosphate, and β-D-galactose-1-phosphate. YcjM catalyzes the phosphorolysis of α-(1,2)-D-glucose-D-glycerate with a kcat = 2.1 s⁻¹, Km = 69 μM, and kcat/Km = 3.1 x 10⁴ M⁻¹ s⁻¹.