In vitro activity of the nisin dehydratase NisB

Abstract

The biosynthesis of several classes of ribosomally synthesized and posttranslationally modified peptides involves dehydration of serine and threonine residues. For class I lantibiotics, thiopeptides, and goadsporin, this dehydration is catalyzed by lanthionine biosynthetic enzyme B (LanB) or LanB-like proteins. Although LanB proteins have been studied since 1992, in vitro reconstitution of their dehydration activity has been elusive. We show here the in vitro activity of the dehydratase involved in the biosynthesis of the food preservative nisin (NisB). In vitro, NisB dehydrated its substrate peptide NisA eight times in the presence of glutamate, ATP, Mg 2+ , and the ribosomal/ membrane fraction of bacterial cell extract. Mutation of 23 highly conserved residues of NisB identified a number of amino acids that are essential for dehydration activity. In addition, these mutagenesis studies identified three mutants, R786A, R826A, and H961A, that result in multiple glutamylations of the NisA substrate. Glutamylation was observed during both Escherichia coli coexpression of NisA with these mutants and in vitro assays. Treatment of the glutamylated substrate with WT NisB results in dehydrated NisA, suggesting that the glutamylated peptide is an intermediate in dehydration. Collectively, these studies suggest that dehydration involves glutamylation of the side chains of Ser and Thr followed by elimination. The latter step has precedent in the virginiamycin resistance protein virginiamycin B lyase. These studies will facilitate investigation of other LanB proteins involved in the biosynthesis of lantibiotics, thiopeptides, and goadsporin.

Capture of micrococcin biosynthetic intermediates reveals C-terminal processing as an obligatory step for in vivo maturation

“…S5). A similar pattern of multiple glutamylations has previously been observed for nonthiopeptide RiPP pathways (22,25); however, in the in vitro reconstituted thiomuracin system, only a monoglutamylated intermediate accumulates when the TclL ortholog (TbtC) is omitted (11). This discrepancy may be due to the different reaction conditions (in vitro versus in vivo) or to an intrinsic difference in how this two-step dehydration process is coordinated in the two biosynthetic systems.…”

“…3). TclK and TclL correspond to the well-characterized Nand C-terminal domains of the Ser/Thr dehydratase NisB from the nisin RiPP pathway (17,22), which allowed us to design mutations in TclK (R181A) and TclL (R49A) that disrupt enzymatic activity and abolish bioactivity in extracts ( Fig. 3 and SI Appendix, Figs.…”

Structural investigation of ribosomally synthesized natural products by hypothetical structure enumeration and evaluation using tandem MS

“…We next applied HSEE to investigate the directionality of catalysis by the nisin dehydratase NisB (33), a prototypical class I lanthipeptide synthetase (19,26). The activity of NisB was recently reconstituted in vitro (33), allowing for a detailed interrogation of its catalytic mechanism. Dehydration by NisB first involves glutamylation of Ser and Thr, followed by more rapid elimination of glutamate to generate the dehydroalanine and dehydrobutyrine residues, respectively.…”

“…7A) was disrupted by site-directed mutagenesis, suggesting that NisB and the cyclase NisC may work in a coordinated fashion (34). However, all nine Ser/Thr residues were dehydrated by NisB in vitro, although the major product was eightfold dehydrated NisA (33). To investigate whether NisB had any selectivity for different dehydration sites on NisA, we performed HSEE analysis on the eightfold dehydrated NisA produced in vitro.…”

Pseudomycoicidin, a Class II Lantibiotic from Bacillus pseudomycoides

“…LanB is required for the selective dehydration of Ser or Thr residues in the LanA core peptide to Dha or Dhb (5). LanC is responsible for the formation of lanthionine rings by linking Cys residues to Dha and Dhb to yield Lan and MeLan, respectively (6).…”