Maturation and secretion of nisin A from Lactococcus lactis
Final Report Abstract
Lantibiotics are ribosomally synthesized and posttranslationally modified antimicrobial peptides that are characterized by the presence of thioether rings, the so-called (methyl)lanthionine rings. In the case of nisin, the prototype of class II lantibiotics, the posttranslational modifications are introduced by two enzymes, NisB, which catalysis the dehydration of serine and threonine residues and NisC, which allows the Michael-type addition of cysteine residues to the dehydrated serine and threonine residues forming the thioether rings. Subsequently, the ABC transporters NisT secretes fully modified nisin into the extracellular space, where the membrane-localized serine protease NisP removes the N-terminal leader peptide resulting in the formation of active nisin (NisA). During this funding period, we could characterized in detail the activity and specificity of secreted NisP demonstrating for the first time that all modification states of nisin are recognized although cleavage occurred at drastically different rates. The ABC transporter NisT could be expressed and purified resulting in a stable and homogenous detergent-solubilized preparation. First reconstitution experiments using synthetic liposome were successfully and will open the way to study the actual secretion process under defined conditions. More important is the fact that the leader peptide of nisin did not modify the basal ATPase activity of the transporter, while fully modified nisin inhibited activity nearly quantitatively. This suggests that not only the leader peptide interacts with the transporter as assumed so far,but also the so-called core peptide. We were also able to reconstitute the maturation complex composed of NisB and NisC in vitro. This allowed us to determine the stoichiometry of the complex as 2: 1: 1 (NisB: NisC: NisA). Interestingly, the complex became highy instable if fully modified nisin was used in the reconstitution experiments suggesting that the formation of the last lanthionine rings represents the trigger for complex disassembly. Finally, this complex was visualized by small angle X-ray scattering (SAXS) revealing for the first time the molecular architecture of the maturation complex. In summary, our biochemical and functional studies provided important insights into the molecular mechanisms by which maturation, secretion and activation of nisin, a model system for lantibiotics, is achieved on the molecular level.
Publications
- (2017) Stoichiometry and structure of a lantibiotic maturation complex. Sci Reports 7 42163
J. Reiners, A. Abts, R. Clemens, S. H. J. Smits & L. Schmitt
(See online at https://doi.org/10.1038/srep42163) - (2017) Substrate specificity of the secreted nisin leader peptidase. NisP Biochemistry 56 4005- 4014
M. Lagedroste, S. H. J. Smits & L. Schmitt
(See online at https://doi.org/10.1021/acs.biochem.7b00524)
