Broadband cryoprobe

All experiments were performed on a Bruker Ascend Avance III 600 MHz system equipped with a broadband cryoprobe (Bruker). Experiments at pH 6 were conducted at 10 or 20°C in 10% D₂O containing 50 mM sodium deuterated acetate buffer, 10 mM NaCl and 100 µM of 3-(trimethylsilyl)propionic 2,2,3,3-d4 acid sodium salt (TMSP) as a chemical shift reference (0 ppm). Experiments at pH 7 were conducted at 10°C in 10% D₂O containing 20 mM sodium phosphate buffer, 10 mM NaCl and 100 µM TMSP as a chemical shift reference (0 ppm). Water signal suppression was achieved using an excitation-sculpting scheme. Thirty-two scans were collected for each spectrum to yield a 15K points-free induction decay.

All experiments were performed on a Bruker Ascend Avance III 600 MHz system equipped with a broadband cryoprobe (Bruker). Experiments were conducted at pH 5.5 or 6.0 at 10 °C in 10% D₂O containing 50 mM sodium deuterated acetate buffer, 10 mM NaCl, and 100 μM 2,2,3,3-²H₄ (trimethylsilyl)propionic acid sodium salt (TMSP) as a chemical shift reference (0 ppm). Water signal suppression was achieved using an excitation-sculpting scheme. Thirty-two scans were collected for each spectrum to yield a 15K-point free induction decay.
To assess the anomerase activity of YhcH, 1 mM 2,3-EN was used as starting substrate in the presence of 20 μM NAD⁺ and 1.5 μM of the hydratase YjhC, which converts 2,3-EN to a mixture of 2,7-AN and α-Neu5Ac (6 ). Anomerases YhcH and NanM were tested at concentrations of 15 μM and 0.5 μM, respectively. We monitored the time course of appearance of shifts specific to H3 axial for both α- and β-Neu5Ac.
To study the anomerization of Neu5Ac catalyzed by divalent cations, 0.6 U/ml of C. perfringens sialidase A (Sigma Aldrich) was used to hydrolyze 1 mM 3’-sialyllactose to lactose and α-Neu5Ac. CoCl₂, NiCl₂, CaCl₂, MgCl₂ were tested at 5 mM and ZnCl₂ was tested at 1 mM. We monitored the time-dependent evolution of peaks corresponding to H3 axial of the alpha-anomer and H3 equatorial of beta-anomer of Neu5Ac.

Human LDH-H (full length and truncated)-6-His proteins for 1D NMR were expressed and purified from E. coli, as described previously. All experiments were performed on an Ascend Avance III 600 MHz system equipped with a broadband cryoprobe (Bruker) following a previously described procedure (16 (link)).
For WaterLOGSY NMR studies, samples were prepared in 10% heavy water containing 50 mM sodium phosphate buffer, pH 7.6, and 100 mM NaCl. The concentration of LDHs was ranging from 15 to 20 μM of monomer. Ligand binding was detected using a WaterLOGSY ephogsygpno.2 avance-version sequence with a 1 s mixing time, 4096 scans were collected at 277 K to yield a 16 K points free induction decay. Water signal suppression was achieved using an excitation-sculpting scheme, and a 50 ms spinlock was used to suppress protein background signals.