Catalytic Dyad in the SGNH Hydrolase Superfamily: In-depth Insight into Structural Parameters Tuning the Catalytic Process of Extracellular Lipase from Streptomyces rimosus.

SrLip is an extracellular enzyme from Streptomyces rimosus (Q93MW7) exhibiting lipase, phospholipase, esterase, thioesterase, and tweenase activities. The structure of SrLip is one of a very few lipases, among the 3D-structures of the SGNH superfamily of hydrolases, structurally characterized by synchrotron diffraction data at 1.75 Å resolution (PDB: 5MAL ). Its crystal structure was determined by molecular replacement using a homology model based on the crystal structure of phospholipase A from Streptomyces albidoflavus (PDB: 4HYQ ). The structure reveals the Rossmann-like 3-layer αβα sandwich fold typical of the SGNH superfamily stabilized by three disulfide bonds. The active site shows a catalytic dyad involving Ser10 and His216 with Ser10-OγH···NεHis216, His216-NδH···O═C-Ser214, and Gly54-NH···Oγ-Ser10 hydrogen bonds essential for the catalysis; the carbonyl oxygen of the Ser214 main chain acts as a hydrogen bond acceptor ensuring the orientation of the His216 imidazole ring suitable for a proton transfer. Molecular dynamics simulations of the apoenzyme and its complex with p-nitrophenyl caprylate were used to probe the positioning of the substrate ester group within the active site and its aliphatic chain within the binding site. Quantum-mechanical calculations at the DFT level revealed the precise molecular mechanism of the SrLip catalytic activity, demonstrating that the overall hydrolysis is a two-step process with acylation as the rate-limiting step associated with the activation free energy of ΔG = 17.9 kcal mol, being in reasonable agreement with the experimental value of 14.5 kcal mol, thus providing strong support in favor of the proposed catalytic mechanism based on a dyad.