Mutants of the Zinc Ligands of Lacticin 481 Synthetase Retain Dehydration Activity but Have Impaired Cyclization Activity

Abstract

Lantibiotics are ribosomally synthesized and post-translationally modified peptide antibiotics. The modifications involve dehydration of Ser and Thr residues to generate dehydroalanines and dehydrobutyrines, followed by intramolecular attack of cysteines onto the newly formed dehydro amino acids to produce cyclic thioethers. LctM performs both processes during the biosynthesis of lacticin 481. Mutation of the zinc ligands Cys781 and Cys836 to alanine did not affect the dehydration activity of LctM. On the other hand, these mutations compromised cyclization activity when investigated with full length or truncated peptide substrates. Mutation of His725, another residue that is fully conserved in lantibiotic cyclases, to Asn resulted in a protein that still catalyzed dehydration of the substrate peptide and also retained cyclization activity, but at a decreased level compared to wild type enzyme. Collectively, these results show that the C-terminal domain of LctM is responsible for cyclization, that the zinc ligands are critical for cyclization, and that dehydration takes place independently from the cyclization activity. Furthermore, these mutant proteins are excellent dehydratases and provide useful tools to investigate the dehydration activity as well as to generate dehydrated peptides for study of the cyclization reaction by wild type LctM. KeywordsLantibiotics; thioether; cyclase; dehydratase; zinc proteinThe lantibiotics are a class of post-translationally modified peptides that have attracted much attention for their potential applications (1,2) exemplified by nisin's commercial use as a food preservative in the dairy industry for over thirty years (3). Understanding the biosynthetic pathways that generate these natural products offers information to improve their production as well as the opportunity to generate novel molecules with promising biological properties. The lantibiotics are produced by Gram-positive bacteria as prepeptides consisting of a leader sequence followed by a propeptide region. This peptide is acted upon by a set of enzymes that successively dehydrate serine and threonine residues in the propeptide to the corresponding dehydroalanine (Dha) and dehydrobutyrine (Dhb) residues, and catalyze the addition of cysteines onto the dehydro amino acids resulting in thioether formation ( Figure 1A). These sulfide crosslinks are called lanthionines (from Ser) or methyllanthionines (from Thr). The class I lantibiotics, like nisin and subtilin, require two distinct enzymes in their biosynthetic pathway to catalyze the dehydration (LanB) and cyclization (LanC) reactions, whereas class II lantibiotics like lacticin 481 have a single enzyme (LanM) that performs both activities ( Figure 1B) (4). The mechanisms by which the LanM enzymes carry out the dehydration and † This work was supported by the National Institutes of Health (GM58822). * vddonk@uiuc.edu. # These authors contributed equally to this work. Figure 1A). Studies conducted in vitro on the bifunctional lacticin 481 and haloduracin synt...

In Vitro Reconstitution and Substrate Specificity of a Lantibiotic Protease

“…Comparing Figures 2a and 2b, it appears that the ionization efficiency of the Cterminal fragment is enhanced upon dehydration and cyclization, but this observation was not further investigated. These results are the first in vitro biosynthesis of authentic lacticin 481 as previous in vitro studies before the availability of LctT150 utilized the commercial protease Lys-C, which produces Δ1-lacticin 481 (11,34,35). As no difference was observed in LctT activity between the linear and cyclized substrates, further studies were continued with linear peptides for simplicity.…”

Pseudomycoicidin, a Class II Lantibiotic from Bacillus pseudomycoides

“…It contains the conserved Zn-binding motifs GXXHGXXG, WCXG, and CHG/CCG (39). The ABC exporter PseT is located immediately downstream of pseA and pseM and harbors an N-terminal double-glycine peptidase domain and the ATP binding site.…”

Lantibiotics and Similar Peptides Produced by and Active on Gram-Positives: Discovery, Development and Perspectives

“…Considerable progress concerning the biochemistry of the modification reactions of lantibiotics has been reported in recent years. In vitro modification systems using LctM from lacticin 481, HalM1/ HalM2 from haloduracin, NisC from nisin and CinM from cinnamycin have been successfully established, and the mechanism and residues involved in the active sites identified (Xie et al 2004;Li et al 2006;You et al 2007;Paul et al 2007;Helfrich et al 2007;McClerren et al 2006). It has been shown, using the lacticin 481 in vitro system, that LctM utilises ATP and Mg 2+ to selectively phosphorylate hydroxy amino acids of the prepeptide before double bond formation (Chatterjee et al 2005).…”