Expanding the Enzyme Repertoire for Sugar Nucleotide Epimerization: the CDP-Tyvelose 2-Epimerase from Thermodesulfatator atlanticus for Glucose/Mannose Interconversion

Epimerases of the sugar nucleotide-modifying class of enzymes have attracted considerable interest in carbohydrate (bio)chemistry for the mechanistic challenges and the opportunities for synthesis involved in the reactions catalyzed. The discovery of new epimerases with an expanded scope of sugar nucleotide substrates used is important to promote mechanistic inquiry and can facilitate the development of new enzyme applications.

The protein standard curve (BioRad molecular mass standards) was used to determine the molecular mass of HisTag-purified TaCPa2E. Dependency of initial rates (s -1 ) on NAD + concentrations (0, 500 and 1000 µM). B.
The first step comprises phosphorylation of the sugar by an anomeric kinase producing glucose-1-phosphate (G1P). The reaction mixture (15 mL) contained 5 mg/mL N-acetylhexosamine 1kinase (NahK), 20 mM ATP, 5 mM MgCl2 and 25 mM D-glucose in MOPS buffer (100 mM, pH 7.5). Incubation at 30 °C was carried out for 16 h until ATP was fully depleted. Phosphorylation progress was monitored with TLC. The second step involves the nucleotidyl transfer from cytidine-5'-triphosphate (CTP) to G1P. The reaction was catalyzed by 1 mg/mL UDP-glucose pyrophosphorylase (UGPase) in the presence of 2 mg/mL inorganic pyrophosphatase (IPPase) and 25 mM CTP. The reaction time was 20 h.   Vivaspin filter tubes (10-30 kDa cut-off) followed by addition of 15% glycerol. Aliquots were stored at -20 °C and -80 °C. A Nanodrop spectrophotometer was used to determine protein concentrations at 280 nm absorbance. Purity and molecular size were shown by SDS-PAGE.

Standard conditions for affinity-tag protein purification
His-tagged enzymes were purified using a HisTrap TM HP column (5 ml Ni Sepharose resin, GE Healthcare Life Sciences), HisTag binding buffer (10 mM HEPES, 500 mM NaCl, 5% (w/w) utilizing His-tag affinity chromatography. The activities of NahK and AcSuSy were tested as described in the literature (10), (11).

CDP-α-D-paratose synthesis (attempted)
The synthesis route is presented in Fig. S1. Dehydration of 1-20 mM CDP-Glc to 2 (see Fig. 1, compound 2) was carried out as described before and the intermediate product used for follow up reactions.

Synthesis under oxic conditions
All experiments were performed with Y. pseudotuberculosis (yp) or S. typhi (st) E1 and E3.
The reaction mixture (0.5-10 mL) contained E1 and E3 in various ratios (1/100-100/1) and concentrations starting from 0.01-80 mg/mL E1 and 0.01-20 mg/mL E3. Various NADH concentrations were tested; either equimolar, higher or lower than the concentration of 2 (see Another approach to improve the product formation involved immobilization of his-tagged ypE3 onto hydrophilic, semi-hydrophilic and hydrophobic resins with Fe(III) coating (EnginZyme) to facilitate the interaction of non-tagged ypE1 with immobilized ypE3 to form an active enzyme complex.

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Resins (30 mg) were equilibrated in 500 µL buffer (5 mM imidazole, 10 mM HEPES, 300 mM NaCl, 5% v/v glycerol). The resins were mixed for 30 min at 30 rpm on an end-over-end rotator and centrifuged for 10 min (6010 g, 4 °C). The supernatant was removed and 500 µL of fresh cell-free extract containing his-tagged ypE3 was combined with the resins. The aforementioned procedure was repeated followed by the replacement of his-tagged ypE3 by non-tagged ypE1 cell-free extract. The binding procedure was performed three times. Coated E1/E3-beads were added in various concentrations to mixtures containing 1 mM of 2 (see Fig. 1), 1.5 mM NADH and 10 µM PMP in MOPS buffer (100 mM, pH 7.5). Reactions were carried out at 30 °C.

Sugar nucleotide isolation
Anion exchange chromatography

Size-exclusion chromatography
Size-exclusion chromatography was used for sodium acetate removal from concentrated product solutions. The Superdex G-10 column (see Methods) was connected to an ÄKTA FPLC system and a 5 mL sample loop. Concentrated samples from anion exchange chromatography were applied directly. Deionized water at a flow rate of 3 mL/min was used for elution and sugar nucleotides were detected by UV-absorption at 254 nm. Product-fractions were pooled and concentrated under reduced pressure (final volume ~10 mL) prior to lyophilization.