Study On The Biosynthesis Mechanism Of Two Ribosomal Peptide Natural Products

The natural products of ribosomally synthesized and posttranslationally modified peptides(RiPPs)are important supplement for natural products apart from Polyketides(PKS)and Non-ribosomal peptides(NRPS).As the antibacterial agent,RiPPs natural products have a narrow antibacterial spectrum,it can avoid broad-spectrum antibiotic off-target effect and the secondary infection which is caused by its resistant strains.It has good prospects for developing clinical drugs.Pantocin A(PA)is a kind of Ri PPs natural product produced from Pantoea agglomerans.It is able to block histidine biosynthesis by inhibiting activity of the L-histidinol phosphate aminotransferase.PA has a good resistance for Erwinia amylovora which causes fire blight,a devastating disease of rosaceous plants such as apple and pear.In 2003,the foreign research group has reported the molecular structure of PA,biosynthetic gene cluster and possible biosynthesis mechanism.However,biochemical characterization of PA biosynthesis has not been reported.In view of the unique molecular structure of PA molecules and their important biological activity.This paper intends to study the key catalytic mechanism of PA biosynthesis.We cloned successfully two key gene,paaA and paaB which participate the biosynthesis of PA molecular from the P.agglomerans genome.paaA and paaB were cloned into pET28 a expression vector respectively.We obtained the expression plasmid and transformed into E.coli BL21 for protein-induced expression and affinity purification.Subsequently,the precursor peptide PaaP chemically synthesized was used as the reaction substrate,PaaA and PaaB proteins were subjected to a bioactivity test.At the same time foreign research group published an article which is named Post-translational Claisen Condensation and Decarboxylation en Route to the Bicyclic Core of Pantocin A.In view of this,we intend to PaaA / B protein crystal structure analysis which laid a foundation for further revealing the catalytic mechanism of PaaA / B.Sactipeptides,formerly known as sactibiotics,are a new class of ribosomally assembled and posttranslational modified natural product peptides.All are assembled from gene encoded precursor peptides that maturate to the corresponding core peptide after introducing at least one thioether bond,bridging the sulfur of a cysteine residue with a highly unreactive a-carbon of an acceptor amino acid.Currently reported Sactipeptides are mostly from aerobic bacteria.However,we found that similar Sactipeptides biosynthetic gene clusters were found in certain anaerobic bacteria such as C.beijerinckii by the bioinformatics analysis.In this study,we used the precursor peptide and the modified enzyme gene in the gene cluster to the pRSFduet-1 vector at the same time,and then expressed in heterologous co-expression in Escherichia coli.The cells were subjected to cell lysis and affinity purification.After electrophoretic detection The purified product was identified by mass spectrometry and was found to differ from the expected molecular weight of 6372.19 by 6.68 Dal.According to the amino acid sequence of SacA,it was presumed that the seven Cys in the precursor peptide SacA could form a disulfide bond in the molecule and reduce the molecular weight by 6Dal.Finally,the modified peptides were analyzed and analyzed by mass spectrometry,and the peptides were formed by the formation of disulfide bonds and the hydrolysis of one amino acid in three groups.The structural identification and activity testing of these new polypeptides is still in progress.