Mestrenova 16

In situ proton NMR analysis was performed in D₂O buffer (50 mM K₂HPO₄/KH₂PO₄, pD 7.5 [pD is the pH meter reading plus 0.4]). The reaction mixture contained 15.3 μM (0.6 mg/ml) TaCPa2E and 4 mM CDP-Glc. Data acquisition was performed at 60°C on a Varian Inova 500-MHz NMR spectrometer (Agilent Technologies, Santa Clara, CA, USA) every 10 min from the enzyme addition. VNMRJ 2.2D software was used for the measurements. ¹H NMR spectra (499.98 MHz) were recorded on a 5-mm indirect-detection pulsed-field-gradient (PFG) probe with presaturation of the water signal by a shaped pulse. The spectra were analyzed using MestReNova 16.0 (Mestrelab Research, SL).

For in situ NMR experiments, the reaction was carried out in D₂O buffer (50 mm K₂HPO₄/KH₂PO₄) titrated to pD 7.6, where pD corresponds to the reading of pH meter + 0.4. The reaction mixture contained 2.7 mm UDP‐GlcA (or UDP‐4‐²H‐GlcA), 91 µm NAD⁺, and 2.4 µm (0.09 mg·mL⁻¹) purified recombinant BcUGAepi. The acquisition was carried out on a Varian INOVA 500‐MHz NMR spectrometer (Agilent Technologies, Santa Clara, CA, USA) every 30 s from the enzyme addition. The VNMRJ 2.2D software was used for the measurements. ¹H‐NMR spectra (499.98 MHz) were recorded on a 5‐mm indirect detection PFG probe with presaturation of the water signal by a shaped pulse. The spectra were analyzed using MestReNova 16.0 (Mestrelab Research, S.L., Santiago de Compostela, Spain).