a, Comparison of protein backbone flexibility using average root mean square fluctuations (RMSF) of Cα atoms calculated per residue along molecular-dynamics simulations of enzymes in the apo conformation. The RMSF is linked to the crystallographic B-factor (B) as follows: \(\text{RMSF}=\surd \left(\frac{3B}{8{\pi }^{2}}\right)\). Red arrows, β-strands; black rectangles, α-helices; yellow rectangles, loops in the X-ray crystal structure of the wild-type LCC (PDB ID 4EB0); dashed lines, positions of catalytic residues. b, Monitoring of key catalytic interatomic distances that characterize the catalytic events occurring during molecular-dynamics simulations of enzymes in complex with the model substrate 2-HE(MHET) 3 . At the right is a representation of the catalytic triad (residues S165 (Ser 165), H242 (His 242) and D210 (Asp 210)) and 2-HE(MHET) 3 , highlighting three relevant interatomic distances (d1, d2, d3). The three graphs show the distributions of these three distances over the first 30 ns of molecular-dynamics simulations of wild-type and ICCG LCC in complex with 2-HE(MHET) 3 (represented as histograms and Gaussian kernel densities), starting from the same initial conformation. Red arrows show changes occurring during the nucleophilic attack of the catalytic serine on the substrate reactive centre; dashed blue lines show hydrogen bonds that assist the catalytic mechanism. The graphs highlight the favoured catalytically productive state adopted by 2-HE(MHET) 3 in variant ICCG. Substantial changes are observed for d1 and d2. Whereas ICCG mainly sampled conformations near the catalytically productive state (average d1 is approximately 3.2 Å; average d2 is approximately 2.8 Å), the wild-type LCC showed a pronounced bimodal distribution with the major conformational population centred on higher distance values, indicating less efficient catalysis. Overall, along the first 30 ns of simulations of these enzymes in complex with 2-HE(MHET) 3 , the average distance separating the substrate cleavage site from the catalytic serine (S165) hydroxyl oxygen was substantially shorter in ICCG than in parental LCC, suggesting that formation of the covalent intermediate during catalysis would be facilitated. c, Occurrence of key hydrogen bonds (HBs) between pairs of catalytic residues. The third and fourth columns show the proportion of snapshots in which an HB interaction is observed between the pairs of catalytic residues S165/H242 and H242/D210 during the first 30 ns of simulations. The higher occurrence of HBs in the ICCG simulation between the S165 hydroxyl oxygen and the catalytic H242 ε nitrogen could assist in the abstraction of the S165 hydroxyl hydrogen by H242, and thus enhance the catalytic performance of this variant.