In vitro CobT activity

CobT assays were performed as described ⁵⁹. Briefly, each reaction (300 μL) contained 30 μg CobT (corresponding to a final enzyme concentration of ~2.6 μM), 2 mM NaMN, 10 mM MgCl₂, and 0.25 mM of either purine or DMB in 50 mM pH 7.5 Tris-HCl buffer. The assay vials were incubated at 30 °C for 30 min before the reactions were terminated by the addition of 15 μL formic acid (≥88%, w/v) and then transferred to a boiling water bath for 1 min. Following neutralizing the pH with 5 M NaOH, precipitated protein was removed by centrifugation at 13,000 × g for 5 min. Lower bases and their respective α-RPs were analyzed by injecting 10 μL samples into an Agilent Technologies 1200 series HPLC system equipped with a diode array detector set to 262 nm. Separation was performed with an Eclipse XDB-C18 column (5 μm pore size, 4.6 mm inner diameter × 250 mm length) at a flow rate of 1 mL per min at 30 °C using 0.1% (v/v) formic acid in water as eluent A and 0.1% (v/v) formic acid in methanol as eluent B. The initial mobile phase and time parameters for the gradient elution were as follows: t = 0, 15% eluent B, 85% eluent A; t = 5 min, 25% eluent B, 75% eluent A; t = 11 min, 85% eluent B, 15% eluent A, t = 15 min, end of run. To obtain material for subsequent MS analysis, semi-preparative HPLC was performed using the method outlined above with minor modification as follows. The injection volume was increased to 100 μL, and a 1-mL fraction containing α-RP [DMB] or α-RP [Purine] was manually collected from the detector outlet once the target peak was detected. Three such injections were performed and the α-RP fractions from each were combined, and then concentrated 10-fold from 3 mL to 300 μL using a Savant ISS110 vacuum dryer (Thermo Fisher). Aliquots (10 μL) of the concentrated α-RP solutions were introduced into an Exactive Plus Orbitrap MS via direct injection. The analytes were ionized using electrospray ionization operated in positive mode and detected via high-resolution MS with a mass range of 200 to 400 m/z ¹⁰.