Lantibiotics are lanthionine-containing antibiotic peptides, which display a number of favorable characteristics. For example, they have no taste or odor, they are tolerant to high heat and acid, and they have stable activity at nanomolar concentrations against antibiotic-resistant pathogens. These attractive qualities are expected to lead to the broad application potentials of lantibiotics (1-4). One of the best known lantibiotics, nisin, which is produced by Lactococcus lactis, has been used as a food preservative for decades. Lantibiotics are divided into two different classes based on the biosynthetic pathway and the enzymes from which they are produced (5, 6). Ribosomally synthesized prepeptide (LanA; "Lan" is an abbreviation for enzymes and/or proteins related to lantibiotic biosynthesis) is inactive and consists of an N-terminal leader peptide and a C-terminal propeptide portion. In class I lantibiotics such as nisin, LanB dehydrates serine and threonine residues of the propeptide, LanC cyclizes dehydrated residues with cysteine, LanT secretes the peptide, and the extracellular protein LanP cleaves off the N-terminal leader peptide. In class II lantibiotics such as nukacin ISK-1, a single enzyme, LanM, catalyzes both dehydration and cyclization, and ABC 2 transporter LanT removes the leader peptide concomitantly with secretion.LanT of the class II lantibiotics is a member of the ABC transporter maturation and secretion (AMS) protein family. Members of this ABC transporter subfamily are involved in the secretion of proteinaceous compounds and contain an additional N-terminal domain involved in the processing of their substrates at the so-called double glycine site at the C terminus of the leader peptide (7-9). There are only a few studies on the N-terminal peptidase domain (PEP) of AMS proteins. The PEPs of LagD, CvaB, ComA, and LctT have been purified and been proven to cleave off the leader peptide of their cognate precursor peptide (7, 9 -11). The PEPs of these proteins have Cys, His, or Asp residues, which are conserved among the papain-like cysteine proteases. Substitution of these residues results in loss of their peptidase activity, demonstrating that PEP also belongs to the cysteine protease family (9 -11). The N-terminal leader peptide sequence of the precursor peptide has been reported to be important for peptidase activity of PEP. Furgerson Ihnken et al. found that the double glycine motif and the ␣-helical structure of the leader peptide are important for proteolysis by PEP of LctT (9). Kotake et al. also demonstrated that PEP of ComA cleaves off the leader peptide after the double glycine site and proposed the presence of hydrophobic patch on the surface of the PEP, which interacts with the hydrophobic face of the helix of the precursor peptide (12). A three-dimensional crystal structure of ComA has been recently clarified, and PEP was