Investigation On The Activity Of Lyase Domain In Class ? Lanthipeptides Synthetase

Class III lanthipeptide is a kind of important natural product of the RiPPs family.During the biosynthesis process,The precursor peptide is dehydrated and cyclized by the Class III lanthipeptides synthetase LanKC and forms mature RiPPs.LanKC is a trifunctional synthetase with an N-terminal lyase domain,a central kinase domain and a putative C-terminal cyclase domain.The sequence alignment revealed that the Nterminal region of LanKC(LanKC-lyase)has a conserved sequence unique to the OspF family.The OspF protein family is a class of phosphothreonine lyases that inhibit the MAPK signaling pathway by dephosphorylation of the phosphokinase of MAPK kinase,thereby affecting many important cell physiology or pathological processes,such as cell growth,differentiation,environmental stress adaptation and inflammatory response.We used heterologous expression,point mutations and in vitro biochemical experiments to study the function and structure-activity relationship of the N-terminal domain of LanKC.The small peptides containing pSer were used for the reaction in vitro,and after LC-MS analysis,we obtained the product of dephosphorylation.The enzymatic kinetic analysis of the enzyme showed that the Km of LanKC-lyase was 50 ?M and the Kcat was 0.78/ S.Further,the seven conserved amino acids in LanKC-lyase were mutated.The results of in vitro reaction showed that the activity of the 7 mutant proteins were all decreased,and the K94 A mutation had the significant effect on the enzyme activity and the effect of R128 A amino acid mutation was insignificant relatively.The mutations of the other five amino acid sites also had a significant effect on the activity of coeKC-lyase cleavage phosphoric acid.The functional difference between the LanKC and OspF family proteins is that the OspF family recognizes a specific phosphorylated threonine of MAPK in the host,while the LanKC proteins recognize all phosphorylated Serine and threonine in precursor peptide.Investigation of the relationship between its function and structure can provide a theoretical basis for the combinatorial biosynthesis of class III lanthipeptides,and finally provide effective candidate molecules for drug screening.