The Relationships Between Structure And Function Of Lantibiotic Bovicin HJ50and Its Leader Peptide

Lantibiotics are ribosomally synthesized antimicrobial polypeptides, containing specialamino acids (such as lanthionine) formed by post-translational modification. The exclusivering structures afford lantibiotics some performances with excellent thermal stability,anti-enzymatic degradation and efficient bactericidal activity. Bovicin HJ50is a novel typeAII lantibiotic produced by Streptococcus bovis HJ50, including two thioether rings (ring Aand ring B) and a characteristic disulfide-bond-containing ring. However, so far, very littleinformation about the correlation between its particular molecular structure and function isknown. Moreover, the role of the leader peptide directing the synthesis of this architecture iswell worth to pay much attention.In order to elucidate the structural features and relationships between the structure andfunction of bovicin HJ50, we prepared bovicin HJ50at milligram level by use of Semi-InVitro Biosynthesis (SIVB) that the propeptide is modified in vivo and the leader peptide isremoved in vitro. After synthesized by this system, the sample was resolved in deturatedacetic acid buffer.2D1H-1H COSY, TOCSY and NOESY, as well as1H-13C HSQC,HSQC-TOCSY and HSQC-NOESY were adopted for the chemical shift assignment andstructural calculation. The results revealed that, in aqueous solution, bovicin HJ50was veryflexibility and the integrate three dimensional structure didn’t existed, which differed muchfrom type AI lantibiotic nisin. The structure of ring A was more convergence, while ring Band ring C were incompact, and regular secondary structure didn’t show up. Circulardichroism (CD) spectrum demonstrated that, bovicin HJ50was randomly coiled in aqueousbuffer but α-helix-like structure was formed in membrane-simulating environment.Take advantage of such structural information, fifty-four mutants and analogs of bovicinHJ50were constructed by means of site-directed mutagenesis and enzymatic treatment to getmore information about the structural trait and functional sites and areas. Antimicrobialactivity analysis displayed that,(1) the charges of N terminus affected very little on activity,but conserved Gly4and aromatic Trp5strongly affect the activity;(2) ring A was a functionalarea, of which Lys11and Asp12were key sites through charge interaction to influence theactivity;(3) ring B residues had minor effect on activity while positive charge of Lys30wasnecessary;(4) hydrophilicity and cyclic structure of ring C was closely related with activity.Besides, this thesis also focused on the secondary structure of bovicin HJ50leaderpeptide to investigate its effects on specific modification and processing. Some associatedmutants were structured and the changes of modification and productivity of mature peptide were checked. The results showed that the areas in relation to secondary structure of theleader peptide played a significant role in bovicin HJ50biosynthesis. We successfullyconstructed seven mutants (F-16A, V-15E, E-14L, E-8P, E-8K, L-7D, L-4K) involved informing secondary structure of the bovicin HJ50leader peptide. MALDI-TOF MS indicatedall the mutants showed2-fold dehydration from their propeptides, assuming the dehydrationis normal. Combining with enzymatic efficient assay, it was found that F-16A, V-15E andL-4K showed very little effect on modification and processing while E-14L and E-8P causedsome changes in modification and E-8K may reduce the processing efficient of BovT150. Inaddition, we found that L-7D resulted in the strongly inhibited removal of leader peptide.In this study, the first solution structure of type AII lantibiotic was resolved. We alsosystematically discussed the relationship between structure and function of bovicin HJ50,which built a solid foundation to investigate the mechanism of its action. All of the abovecould be referred by the study of the same type lantibiotics.