[Individual catalytic activity of two functional domains of bovicin HJ50 synthase BovM].

OBJECTIVE: To reconstitute the in vitro catalytic activity of the individual dehydratase or cyclase domain of bifunctional bovicin HJ50 synthase BovM, and lay a foundation for the further investigation of catalytic mechanism of class II lantibiotic synthase LanM. METHOD: The truncated proteins of BovM containing the N-terminal dehydratase domain or C-terminal cyclase domain were expressed in E. coli and purified. Substrate BovA, the precursor of bovicin HJ50, was incubated with these truncated BovM proteins in in vitro reaction system. The antimicrobial activity assay and MALDI-TOF MS analysis were used to monitor the dehydratase or cyclase activity of these truncated proteins. Meanwhile, the synergistic activities of both truncated proteins were tested in vivo and in vitro. RESULTS: The N- and C-terminal domains of BovM possessed dehydration and cyclization activity respectively. However, no synergistic activity was detected between these two functional domains. CONCLUSION: The individual functional domains of BovM could execute their corresponding functions independently, but the intactness of BovM was important for its full modification activity.

Complete genome sequence of Lactococcus lactis S0, an efficient producer of nisin.

Lactococcus lactis S0 is a nisin Z-producing strain isolated from milk, and the nisin production of the strain can reach 4000 IU/ml under fermenting condition. Here, we present the complete genome sequence of L. lactis S0 which includes a single circular chromosome.

[Semi-in vitro biosynthesis of cryptic lanthipeptide CLA 124 from Streptomyces clavuligerus].

OBJECTIVE: To obtain the cryptic lanthipeptide from Streptomyces clavuligerus by semi-in vitro biosynthesis that is a novel method for mining lanthipeptides resource from Streptomyces. METHODS: The core peptide of cryptic lanthipeptide was modified in E. coli by nisin modification system, and purified by affinity chromatography and High Performance Liquid Chromatography (HPLC). After the leader peptide was removed, the core peptide was obtained and its dehydration and cyclic structure were analyzed by MALDI-TOF MS and tandem MS. RESULTS: A novel lanthipeptide named CLA 124 with 4-fold dehydration 2 thioether bridges and one disulfide bridge was produced. CONCLUSION: Cryptic lanthipeptides from Streptomyces could be produced by semi-in vitro biosynthesis.

Dissecting the catalytic and substrate binding activity of a class II lanthipeptide synthetase BovM.

LanM proteins are the synthetases of the class II lanthipeptides, which are responsible for lanthionine or methyllanthionine formation in lanthipeptides. LanMs are bifunctional enzymes with N-terminal dehydratase and C-terminal cyclase domains. However, the catalytic and especially the substrate binding function of LanM are not fully investigated. In this study, we analyzed the function of conserved residues of BovM, which is the synthetase of lanthipeptide bovicin HJ50, with alanine substitution method. Mass spectrometry (MS) and surface plasmon resonance (SPR) analyses showed six hydrophilic residues (e.g. Asp247) were involved in the dehydration activity of BovM and four hydrophobic residues (e.g. Ile254) were responsible for the substrate binding of BovM. In addition, a conserved Asp155 was proposed to be general base in the elimination of phosphates during the dehydration reactions. This research of BovM shed a light on the catalytic and substrate binding mechanism of LanM proteins.

Bovicin HJ50-like lantibiotics, a novel subgroup of lantibiotics featured by an indispensable disulfide bridge.

Lantibiotics are ribosomally-synthesized and posttranslationally modified peptides with potent antimicrobial activities. Discovery of novel lantibiotics has been greatly accelerated with the soaring release of genomic information of microorganisms. As a unique class II lantibiotic, bovicin HJ50 is produced by Streptococcus bovis HJ50 and contains one rare disulfide bridge. By using its precursor BovA as a drive sequence, 16 BovA-like peptides were revealed in a wide variety of species. From them, three representative novel lan loci from Clostridium perfringens D str. JGS1721, Bacillus cereus As 1.348 and B. thuringiensis As 1.013 were identified by PCR screening. The corresponding mature lantibiotics designated perecin, cerecin and thuricin were obtained and structurally elucidated to be bovicin HJ50-like lantibiotics especially by containing a conserved disulfide bridge. The disulfide bridge was substantiated to be essential for the function of bovicin HJ50-like lantibiotics as its disruption eliminated their antimicrobial activities. Further analysis indicated that the disulfide bridge played a crucial role in maintaining the hydrophobicity of bovicin HJ50, which might facilitate it to exert antimicrobial function. This study unveiled a novel subgroup of disulfide-containing lantibiotics from bacteria of different niches and further demonstrated the indispensable role of disulfide bridge in these novel bovicin HJ50-like lantibiotics.