Study Of The Interaction Mechanism Of Glycine Cleavage System In One-carbon Synthetic Pathway

Currently,with the increased consumption of first-generation fossil energy and second-generation sugar-based feedstocks,one-carbon(C1)metabolism,which is a production method that using cheap and easily available CO₂ as a raw material for biosynthesis to produce high value-added products,has gradually emerged and become a research hotspot.The reduced glycine pathway is one of the most promising biosynthetic pathways due to its distance from the metabolic center and its ability to maintain a reasonably high chemical kinetics throughout the reaction.However,the low efficiency of glycine synthesis in the reduced glycine pathway is the main bottleneck in industrial application.The enzyme that dominates the synthesis of glycine is the glycine cleavage system.The glycine cleavage system is a multi-enzyme complex,and it is composed of the shuttle protein H and the other three catalytic proteins P,T,and L.The reaction is reversible.However,glycine cleavage system catalyzes the cleavage of glycine more than the synthesis of glycine.This is the main reason for the insufficient efficiency of glycine synthesis.Through computational analysis,we have studied the catalytic mechanism between the proteins of the glycine cleavage system and confirmed the results by the means of mutation experiments,offering a fundamental theory for the directed evolution of the glycine cleavage system.The research includes the following aspects:1.Molecular dynamic simulations of protein H induced by protein T release aminomethyl moiety and experiment verification in a key reaction step.The protein H in the glycine cleavage system will embrace the lipoamide arm in the hydrophobic cavity for protection.Only the induction of protein T could release the lipoamide arm by triggering the conformational change of protein H and participating in the following reaction.Here,we first examined the induction of conformational changes in protein H by protein T using molecular dynamics(MD)simulations and identified key steps and amino acid residues involved in the release of the aminomethyl lipoate arm.Umbrella sampling was used to analyze the conformational changes of proteins H and T.Mutagenesis experiments of Ser-67 were carried out to confirm a key amino acid residue identified for the release of the aminomethyl lipoate arm.The results reveal important molecular details about a key reaction step of glycine cleavage system and pave the way for a purposeful manipulation of a fundamentally important enzyme complex in C1 metabolism.2.In the glycine cleavage system,pyridoxal phosphate(PLP)assists protein H to complete the self-protection process.In the reverse reaction process of the glycine cleavage system,the lipoic acid arm of the protein H is released into the T-protein and reacted to form lipoamide.After that,how does the lipoamide enter into the hydrophobic cavity of the protein H to form a stable embrace structure?We carried out many speculations and carried out atomic-level molecular dynamic simulations to verify these speculations to answer the question.We first speculated that this self-protection process is the reverse process of the release process,which is also induced by protein T.However,the simulation results show that the lipoamide arm cannot enter into the hydrophobic cavity of the protein H induced completely by the T-protein.Afterward,inspired by the experimental observations that stand-alone protein H enabled glycine cleavage and glycine synthesis,we speculated that the protein H alone could complete the self-protection process.However,after molecular dynamic simulations of protein H,the stand-alone protein H also cannot complete the self-protection process thoroughly.Based on the inspiration of protein H with the function of protein P,we linked the Lys-11 of H-protein to PLP covalently.Unexpectedly,we found that the protein H can complete the self-protection process thoroughly this time.Umbrella sampling was used to analyze the energy barrier in the process,and two energy barriers were found in the self-protection process.It appears that protein H only crosses the first energy barrier by itself or by the induction of protein T.The second energy barrier can be crossed with the assistance of PLP.To validate the conjecture,we mutated Lys-11 to Ala,the enzyme activity of the overall glycine cleavage system increased significantly.It suggests that the efficiency of stand-alone protein H is much lower than that of protein P.Once protein H does not serve as the functional enzyme and only serves as the shuttle protein to link the active sites of the other three functional proteins,the overall reaction activity is significantly improved.The discovery of the new function of PLP also provides a practical new way to modify the glycine cleavage system.3.QM/MM study on the reaction mechanism of protein T capturing ammonia in the glycine cleavage system.In the three-step cyclic reaction of the glycine cleavage system,the catalytic mechanism of the decarboxylation/carboxylation performed by protein P and the oxidation/reduction reaction performed by protein L have been studied thoroughly.The reaction mechanism of ammonia transfer/capture performed by protein T has not been reviewed yet.To have a further understanding of the catalytic mechanism of protein T for ammonia capturing and then having insight into the overall reaction mechanism of the glycine cleavage system,we studied the process of protein T capturing ammonia at Quantum-Mechanics/Molecular-Mechanics(QM/MM)level.We first built the initial reaction system based on the crystallized structure and carried out molecular dynamic simulations.After that,we clustered the conformations in the molecular dynamics trajectory of the initial reaction system and obtained the representative conformation with the largest number of clusters.Geometry optimization at the QM/MM level was used to improve the structure until the convergence reached.Then we guessed the electron transfer process based on the convergence conformation.The key of the process is the nucleophilic addition between the ammonium and the methylene.Now,we are searching for a transition state.We plan to draw the reaction path analysis(Intrinsic Reaction Coordinate,IRC)curve of the reaction process in the next step.Our study also provides further insights into the bioconversion of organic ammonia.