Discovery and in vitro biosynthesis of haloduracin, a two-component lantibiotic

Abstract

Lantibiotics are ribosomally synthesized peptides that undergo posttranslational modifications to their mature, antimicrobial form. They are characterized by the unique amino acids lanthionine and methyllanthionine, introduced by means of dehydration of Ser͞Thr residues followed by reaction of the resulting dehydro amino acids with cysteines to form thioether linkages. Two-component lantibiotics use two peptides that are each posttranslationally modified to yield two functionally distinct products that act in synergy to provide bactericidal activity. By using genetic data instead of isolation, a two-component lantibiotic, haloduracin, was identified in the genome of the Gram-positive alkaliphilic bacterium Bacillus halodurans C-125. We show that heterologously expressed and purified precursor peptides HalA1 and HalA2 are processed by the purified modification enzymes HalM1 and HalM2 in an in vitro reconstitution of the biosynthesis of a two-component lantibiotic. The activity of each HalM enzyme is substratespecific, and the assay products exhibit antimicrobial activity after removal of their leader sequences at an engineered Factor Xa cleavage site, indicating that correct thioether formation has occurred. Haloduracin's biological activity depends on the presence of both modified peptides. The structures of the two mature haloduracin peptides Hal␣ and Hal␤ were investigated, indicating that they have similarities as well as some distinct differences compared with other two-component lantibiotics.lanthionine ͉ dehydroalanine ͉ antibiotic

In Vitro Reconstitution and Substrate Specificity of a Lantibiotic Protease

“…Non-conservative mutations of residues in the P1′-P5′ positions (K1A, G3I, G5H), however, did not affect proteolysis by His 10 -LctT150, and several chimeric peptides containing multiple simultaneous mutations in these positions (eg P1′: Asn, Trp, Gly; P2′: Lys, Arg, Asn; P3′: Ser, Trp, Gly, Lys (Table 3 and Figure 5)) were likewise processed. Thus, the residues C-terminal to the cleavage site are not important, which may also explain why some two-component lantibiotic biosynthetic gene clusters contain only a single LanT protein despite dramatic differences between the two propeptides of their substrates (12,48). A possible exception to the high promiscuity with respect to the residues C-terminal to the cleavage site may be a branched residue at the P1′ position because the nisin chimera with an Ile at this position was not accepted.…”

“…It may act in a protective role for the producing organism, as its removal is required for bioactivity (10)(11)(12)(13)(14)(15). The leader peptide could also serve as a recognition motif for the transport machinery because non-lantibiotic peptides attached to leader peptides are secreted (16,17), or as a scaffold for the lantibiotic synthetases (11,(18)(19)(20).…”

Fundamental functionality: recent developments in understanding the structure–activity relationships of lantibiotic peptides

“…A Ser26Ala mutant was also produced and was found to retain activity. 8,20,46 Ring A of haloduracin-b was found to have a small effect on activity, with a moderate reduction of activity in the mutant. Rings C and D were both shown to be important, as mutations resulted in very little remaining activity.…”

“…[43][44][45] HALODURACIN Haloduracin is a two-component lantibiotic isolated from Bacillus halodurans consisting of a globular a-peptide resembling the class B lantibiotics and an elongated b-peptide similar to class A. 46,47 Haloduracin-a has one cysteine, one lan and two melan rings. Haloduracin-b contains one lan and three melan rings as well as Structure-activity relationships of lantibiotic peptides AC Ross and JC Vederas three Dhbs.…”

Genetic characterisation and heterologous expression of leucocin C, a class IIa bacteriocin from Leuconostoc carnosum 4010

“…On the other hand, type II lantibiotics, such as lacticin 481, mersacidin, and actagardin are modified by only one bifunctional enzyme, LanM (Siezen et al 1996). Two-peptide lantibiotics like lacticin 3147 and haloduracin are also included in this class (Ryan et al 1996;McClerren et al 2006). Type III lantibiotics were described as lanthionine-containing peptides without antimicrobial activity.…”