Nucleotide Sugar Hydrolysis Assay

The ultra-pure UDP-Glc, UDP-Gal, and UDP-GlcA as well as UMP/CMP-Glo™ Glycosyltransferase Assay Kit were purchased from Promega Corporation, Madison, WI, USA. The measurement of the donor specificity of the recombinant PssA, determined as the hydrolysis of the nucleotide sugar derivative, was carried out by incubating a given amount (0.1–5 μg) of recombinant His6-PssA in the reaction buffer: 50 mM HEPES, 100 mM NaCl, 5 mM MgCl₂, 0.1% Tween-20 [23 (link)] with a given sugar substrate (100 μM) for 4 h at 28 °C. Control mixtures contained no enzyme. The hydrolysis reaction was stopped by adding UMP/CMP Detection Reagent^TM in a 1:1 ratio (25 μL:25 μL). After mixing, the plate was incubated at room temperature for 1 h. Then the level of luminescence was measured with Synergy H1 multi-detection reader (BioTek, Agilent Technologies, Santa Clara, CA, USA). The intensity of emitted light (luciferase reaction) is proportional to the amount of free UMP produced in the UDP-sugar hydrolysis reaction. Each reaction was performed in a separate well of a 96-well plate in two technical repeats.