Integrating protein contact networks for the engineering of thermostable lipase A

In the field of industrial biocatalysis, the rapid advancement of enzyme functional evolution necessitates new theories and computational methods to achieve target functions with fewer iterations. This study identified key residues affecting enzyme stability by constructing the protein contact network (PCN) of Lipase A. Comparing the PCNs of the wild-type (WT) and the 6B variant revealed that changes in residue interactions and node properties (e.g., degree and betweenness centrality (BC)) positively impacted stability. Using thresholds for degree and BC, 25 candidate sites were screened, and 11 out of 18 single-point mutation designs improved thermal stability. Mutations were divided into three groups (M1, M2, M3) based on network communities and contributions, followed by iterative combinations. M1, containing five mutations distributed across four communities, increased the melting temperature (T_m) by 14.61 °C, close to the predicted 13.97 °C, demonstrating a linear additive effect. In M2, three new mutations resulted in a non-linear additive effect, with a ΔT_m of 17.58 °C (Expected ΔT_m = 18.93 °C). In contrast, the three new mutations in M3 destabilized the enzyme (Observed ΔT_m = 15.94 °C vs Expected ΔT_m = 19.92 °C). Molecular dynamics simulations showed that polar edge nodes enhanced network connectivity, while proline mutations rigidified flexible regions, improving stability. Conversely, M3 mutations disrupted α-helix stability by increasing the dihedral angle fluctuations of residue Y161, might to a stability-activity trade-off. The PCN provides valuable insights for developing efficient and precise design strategies.