title: Substrate recognition and specificity of the NisB protein, the lantibiotic dehydratase involved in nisin biosynthesis.
abstract: Nisin is a posttranslationally modified antimicrobial peptide containing the cyclic thioether amino acids lanthionine and methyllanthionine. Although much is known about its antimicrobial activity and mode of action, knowledge about the nisin modification process is still rather limited. The dehydratase NisB is believed to be the initial interaction partner in modification. NisB dehydrates specific serine and threonine residues in prenisin, whereas the cyclase NisC catalyzes the (methyl)lanthionine formation. The fully modified prenisin is exported and the leader peptide is cleaved off by the extracellular protease NisP. Light scattering analysis demonstrated that purified NisB is a dimer in solution. Using size exclusion chromatography and surface plasmon resonance, the interaction of NisB and prenisin, including several of its modified derivatives, was studied. Unmodified prenisin binds to NisB with an affinity of 1.05 ± 0.25 μm, whereas the dehydrated and the fully modified derivatives bind with respective affinities of 0.31 ± 0.07 and 10.5 ± 1.7 μm. The much lower affinity for the fully modified prenisin was related to a >20-fold higher off-rate. For all three peptides the stoichiometry of binding was 1:1. Active nisin, which is the equivalent of fully modified prenisin lacking the leader peptide did not bind to NisB, nor did prenisin in which the highly conserved FNLD box within the leader peptide was mutated to AAAA. Taken together our data indicate that the leader peptide is essential for initial recognition and binding of prenisin to NisB.
authors.affiliation: Institute of Biochemistry, Heinrich Heine University Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany.
authors.forename: Antonino
authors.initials: A
authors.lastname: Mavaro
authors.affiliation: Institute of Biochemistry, Heinrich Heine University Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany.
authors.forename: André
authors.initials: A
authors.lastname: Abts
authors.affiliation: Institute of Biochemistry, Heinrich Heine University Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany.
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Bakkes
authors.affiliation: BiOMaDe Technology Foundation, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Gert N
authors.initials: GN
authors.lastname: Moll
authors.affiliation: Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, Zernike Institute for Advanced Materials and the Kluyver Centre for the Genomics of Industrial Microorganisms, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands.
authors.forename: Arnold J M
authors.initials: AJM
authors.lastname: Driessen
authors.affiliation: Institute of Biochemistry, Heinrich Heine University Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany.
authors.forename: Sander H J
authors.initials: SHJ
authors.lastname: Smits
authors.affiliation: Institute of Biochemistry, Heinrich Heine University Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany. Electronic address: lutz.schmitt@hhu.de.
authors.forename: Lutz
authors.initials: L
authors.lastname: Schmitt
mesh_terms: D000595:Amino Acid Sequence; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry*; D001430:Bacteriocins / Q000096:biosynthesis* / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D019281:Dimerization; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D009561:Nisin / Q000096:biosynthesis*; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D012542:Scattering, Radiation; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance
chemical_list: D023181:Antimicrobial Cationic Peptides; D001426:Bacterial Proteins; D001430:Bacteriocins; D008565:Membrane Proteins; C079126:NisB protein, Lactococcus lactis; D010455:Peptides; D009561:Nisin