Discovery of a Kojibiose Phosphorylase in Escherichia coli K-12

The substrate profiles for three uncharacterized enzymes (YcjM, YcjT, and YcjU) that are expressed from a cluster of 12 genes ( ycjM-W and ompG) of unknown function in Escherichia coli K-12 were 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 ( k_cat = 1.1 s^-1, K_m = 1.05 mM, and k_cat/ K_m = 1.12 × 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 ( k_cat = 21 s^-1, K_m = 18 μM, and k_cat/ K_m = 1.1 × 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 k_cat = 2.1 s^-1, K_m = 69 μM, and k_cat/ K_m = 3.1 × 10⁴ M^-1 s^-1.