Figure 6. The free energy profile and optimized structures of important transition states (TSs) for hydrolysis reaction in NocTE-DSub (A) and NocTE-LSub (B) systems. The important residues (D1806, H1808 and H1901), DSub and LSub were colored in gray, blue and pink respectively.
The special spatial location of WatA revealed the competition between epimerization and hydrolysis in NocTE-LSub system. WatA was not only able to mediate the proton transfer between H1901 and the Cα atom, but also had the potential to directly participate in the hydrolysis of LSub. When H1901 captured the proton H1 of WatA, the generated nucleophile could choose to reacquire a proton from Cα atom to complete the deprotonation step, or attack the C1 atom to form the hydrolytic intermediate. However, the distance between the O atom of WatA and the Hα atom (2.42 Å) was closer than that for the C1 atom (3.29 Å), so WatA is more advantageous in acquiring the proton Hα (Figure S11). In addition, the Hpg residue in the C-terminal of LSub caused certain steric hindrance and the unreasonable orientation in the hydrolysis process prevented WatA from forming a stable hydrogen bond with H1808, unbeneficial for maintaining low-energy conformations. These structural disadvantages seriously hindered WatA from getting close to C1 atom directly, making it difficult to promote the hydrolysis reaction.
Aimed to assess the difficulty of direct hydrolysis of LSub, we obtained the energy profile of hydrolysis reaction starting from the initial conformation for previous epimerization study (Figure 6B, Table S8). Different from the energy profile of DSub hydrolysis, the rate-limiting step for LSub hydrolysis was the nucleophilic attack step of WatA towards the C1 atom rather than the step of breaking the covalent bond between S1779 and substrate, which could be well explained by structural disadvantages mentioned above. In addition, the energy barrier for LSub hydrolysis was 11.6 kcal/mol higher than that for the deprotonation step of epimerization, indicating the early-stage epimerization was essential for NocTE catalysis and further accounting for stereochemical selectivity.