Investigation Of Catalytic Mechanism Of SurE: A Member Of New Cyclase Family In Non-Ribosomal Peptide Synthases

Cyclic peptides are one of highly medicinal functional molecules.The obstacle of its chemical synthesis lies in the accurate coupling of the ring closing sites of linear peptide precursors.It was found that a new family of NRPS peptide cyclase,called PBP-like thioesterase,represented by Sur E,could be used as an independent protein to catalyze the head to tail macrocyclization of liner peptides with highly stereo-specificity(N-terminus with L-type and C-terminus with D-type),which is different from the TE domain fixed at the end of NRPS assembly line.It has high potential to applied as a new biocatalytic components.However,the catalytic mechanism of this novel family has not been reported,which limits its further application in catalysis.This work investigated the uploading-recognition-catalytic process of the substrate,starting with the crystal structure of Sur E,which is performed with molecular dynamics simulation and quantitative calculation as the main research methods,combining with the reported in vitro experimental data to build a series of computational models.In the course of theoretical research,we found that the change of C-terminal amino acid configuration on the substrate significantly affected the uploading efficiency of the substrate due to the steric effect.It was also found that the R446 plays an important role in recognition.The exclusively formation of bidentate hydrogen bonds between guanidine group and substrate’s N-terminus with L-type amino acid helps to maintain the pre-organized stated of substrate,enabling Sur E with the preference of N-terminal L-type.Each amino acids forming the essential catalytic hydrogen bond network(Y154-K66-N156-substrate)in Sur E promoted the binding affinity by MM-GBSA free energy decomposition calculation.The maintenance of the catalytic hydrogen bond networks also enables Sur E with the preference of C-terminus D-type.Based on the catalytic hydrogen bond networks during the MD simulations,we proposed the Sur E catalytic mechanism,verified by ONIOM(QM/MM)calculations.The proton chain between Y154 and K66 reacted as a deprotonation pathway during macrocyclization,which is a concerted reaction with an energy barrier of 16.9 kcal/mol,while 2.1 kcal/mol energy is released to form the acyl-tetrahedron intermediate.Finally,the proton on K66 is returned to S63 to complete the catalytic cycle and release the product.This process requires crossing the energy barrier of 4.6 kcal/mol and releasing22.2 kcal/mol energy.Mutation experiments supported our proposed mechanism,revealing that the function of the catalytic quadruplet of Sur E(S63-K66-Y154-N156).The rational design of substrates containing non-natural amino acid fragments reveals the well substrate tolerance of Sur E.This work,as the first report on the catalytic mechanism of PBP-like TEs family,elucidated the whole process mechanism of Sur E and revealed the function of key residues.It was an inspiring study for the following family of such novel cyclases and provided theoretical guidance for the targeted modification of Sur E as well as the development of novel cyclic peptides.