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.
Highlights
Applying the previously described modification enzyme co-purification binding assay (Khusainov et al, 2011) we showed that the NisB mutants Y80F and H961A are still able to bind precursor nisin (Figure S1)
The effects of the mutations that we observed can be classified into three groups: (1) mutations that resulted in a wild type extent of dehydration and secretion, (2) mutations that resulted in non-secreted peptides with intermediate dehydration patterns, and (3) mutations that resulted in non-secreted and unmodified precursor nisin
NisT has been demonstrated of being capable of exporting unmodified precursor nisin in the absence of NisB (Kuipers et al, 2004)
Summary
The rings contain post-translationally introduced thioether-bridged amino acids, so called lanthionines. The leader peptide is a recognition signal for the modification enzymes NisB and NisC (Xie et al, 2004; Mavaro et al, 2011; Khusainov et al, 2013a) and the transporter NisT (van der Meer et al, 1994). It keeps the fully modified precursor nisin inactive (Kuipers et al, 1993b; van der Meer et al, 1994). Analysis of truncated nisin variants has shown that the presence of at least the three Nterminal rings ABC is necessary for nisin variants to exert some antimicrobial activity (Chan et al, 1996)
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