After immobilized metal affinity chromatography, SyrB1 was buffer exchanged into RXN buffer three times using Amicon™ Ultra 50 kDa MWCO centrifugal filter units (Millipore Sigma). To attach the Ppt cofactor, SyrB1 (100 µM) was incubated with coenzyme A (1 mM, Sigma: cofactor for acyl transfer), MgSO4 (5 mM, Sigma: BioReagent), and phosphopantetheinyl transferase Sfp (5 µM) for 90 minutes at room temperature. The reaction mixture was concentrated using the same centrifugal filter units and then diluted to ~8 mL with reaction buffer. After filtering, the protein was loaded into the superloop of an AKTA Pure protein purification system. The same elution method as described for the SyrB2 proteins was performed except that the flow rate was adjusted to 0.8 mL/min. Relevant fractions from size-exclusion chromatography were pooled and concentrated using centrifugal filter units. To append threonine to the protein, SyrB1-Ppt (100 µM) was incubated with MgSO4 (5 mM), ATP (10 mM, Sigma: Grade I, ≥99%, from microbial), and L-threonine (10 mM, Sigma: reagent grade, ≥98% (HPLC)) in RXN buffer for 30 min at room temperature.