The substrate selectivity of the two homologous SGNH hydrolases from Streptomyces bacteria: Molecular dynamics and experimental study.

Two extracellular enzymes of the SGNH hydrolase superfamily reveal highly homologous 3D structures, but act on different substrates; one is a true phospholipase A1 from Streptomyces albidoflavus (SaPLA1, EC: 3.1.1.32, PDB code: 4HYQ), whereas the promiscuous enzyme from Streptomyces rimosus (SrLip, EC: 3.1.1.3, PDB code: 5MAL) exhibits lipase, phospholipase, esterase, thioesterase, and Tweenase activities. To get insight into binding modes of phospholipid and triglyceride substrates in both enzymes and understand their chain-length preferences, we opted for computational approach based on in silico prepared enzyme-substrate complexes. Docking procedure and molecular dynamics simulations at microsecond time scale were applied. The modelled complexes of SaPLA1 and SrLip enzymes revealed substrate accommodation: a) the acyl-chain attached to sn-1 position fits into the hydrophobic pocket, b) the acyl-chain attached to sn-2 position fits in the hydrophobic cleft, whereas c) the sn-3 bound acyl chain of the triglyceride or polar head of the glycerophospholipid fits into the binding groove. Moreover, our results pinpointed subtle amino acid differences in the hydrophobic pockets of these two enzymes which accommodate the acyl chain attached to sn-1 position of glycerol to be responsible for the chain length preference. Slight differences in the binding grooves of SaPLA1 and SrLip, which accommodate the acyl chain attached to sn-3 position are responsible for exclusive phospholipase and both phospholipase/lipase activities of these two enzymes, respectively. The results of modelling correlate with the experimentally obtained kinetic parameters given in the literature and are important for protein engineering that aims to obtain a variant of enzyme, which would preferably act on the substrate of interest.