| Lanthipeptides are the earliest discovered Ri PPs with antibacterial,antifungal,antiviral and antiallodynic activities.The thioether amino acids lanthionine(Lan)or methyllanthionine(Me Lan)is the typical moiety of lanthipeptide.These residues are installed via a two-step biosynthetic process catalyzed by Lan synthetase.Ser/Thr residues in the core peptide are first dehydrated to Dha and Dhb,respectively.Then,Cys thiols are added to the unsaturated amino acids through a Michael-type addition to form Lanthionine.Based on the biosynthetic machinery responsible for thioether rings formations,lanthipeptides can be subdivided into four different classes,class I-IV.Previously,we discovered a novel lanthipeptide,named lexapeptide,which contains a lanthionine ring and an Avi Me Cys ring.According to the bioinformatics analysis and genetics experimental prediction,we found that the key tailoring enzymes Lxm X and Lxm Y exhibit no similarity with any known Lan synthetases.We here assign it as a novel Lan synthetase class,class V,containing Lxm K,Lxm Y and Lxm X.However,there is no experiment result to support our assumption.Moreover,the specific functions of Lxm X or Lxm Y are not clear either.In this study,the precursor and tailoring enzymes are individually overexpressed and co-expressed in order to study the dehydration and cyclization via in vitro reaction and in vivo reconstitution.The linear intermediates were found unstable in vivo.In the experiment,the core peptide of lexapeptide cannot be modified without leader region.Lexapeptide B biosynthesis was successfully reconstituted in E.coli BL21(DE3)through co-expression of five core genes,the precursor peptide lxm A,and four postmodified enzymes,lxm KDXY.These results provided evidence that Lxm K,Lxm Y and Lxm X co-catalyzed biosynthesis of lanthionine,which reveals that class V Lan synthetases are composed of Lxm K,Lxm Y and Lxm X.The Lxm X crystal and the selenomethionyl protein Lxm X’(S129/189M)crystal were obtained successfully at the resolution of 2.05? and 2.9?,respectively.Lxm X is one of the key enzymes in thioether ring formation of lexapeptide.The results provide a basis on Lxm X structure analysis and the research regarding its function and catalytic mechanism.We also initially explored possible resistance genes in lexapeptide gene cluster via gene knockout and heterologous expression.lxm T,lxm I and orf1-8 was studied and orf7 may be a potential resistance gene. |
