title: Identification of essential amino acid residues in the nisin dehydratase NisB.
abstract: Nisin is a posttranslationally-modified antimicrobial peptide that has the ability to induce its own biosynthesis. Serines and threonines in the modifiable core peptide part of precursor nisin are dehydrated to dehydroalanines and dehydrobutyrines by the dehydratase NisB, and subsequently cysteines are coupled to the dehydroamino acids by the cyclase NisC. In this study, we applied extensive site-directed mutagenesis, together with direct binding studies, to investigate the molecular mechanism of the dehydratase NisB. We use a natural nisin-producing strain as a host to probe mutant-NisB functionality. Importantly, we are able to differentiate between intracellular and secreted fully dehydrated precursor nisin, enabling investigation of the NisB properties needed for the release of dehydrated precursor nisin to its devoted secretion system NisT. We report that single amino acid substitutions of conserved residues, i.e., R83A, R83M, and R87A result in incomplete dehydration of precursor nisin and prevention of secretion. Single point NisB mutants Y80F and H961A, result in a complete lack of dehydration of precursor nisin, but do not abrogate precursor nisin binding. The data indicate that residues Y80 and H961 are directly involved in catalysis, fitting well with their position in the recently published 3D-structure of NisB. We confirm, by in vivo studies, results that were previously obtained from in vitro experiments and NisB structure elucidation and show that previous findings translate well to effects seen in the original production host.
authors.affiliation: Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen Groningen, Netherlands.
authors.forename: Rustem
authors.initials: R
authors.lastname: Khusainov
authors.affiliation: Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen Groningen, Netherlands.
authors.forename: Auke J
authors.initials: AJ
authors.lastname: van Heel
authors.affiliation: Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen Groningen, Netherlands.
authors.forename: Jacek
authors.initials: J
authors.lastname: Lubelski
authors.affiliation: LanthioPharma Groningen, Netherlands.
authors.forename: Gert N
authors.initials: GN
authors.lastname: Moll
authors.affiliation: Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen Groningen, Netherlands ; Kluyver Centre for Genomics of Industrial Fermentation Groningen, Netherlands.
authors.forename: Oscar P
authors.initials: OP
authors.lastname: Kuipers
keywords: NisB; NisC; dehydratase; lanthionine; lantibiotics; mechanism; nisin; posttranslational modifications