Mechanistic investigation of HalM2, one of two bifunctional enzymes involved in haloduracin biosynthesis

Lantibiotics are ribosomally synthesized peptides that undergo post‐translational modifications to their mature, antimicrobial form. They are characterized by the presence of the unique amino acid lanthionine, which is introduced via dehydration of Ser/Thr residues followed by reaction of the resulting dehydro amino acids with cysteines to form thioether linkages. Two‐component lantibiotics utilize two peptides that are each post‐translationally modified to their active form and act in synergy to provide bactericidal activity. Using genetic data instead of isolation, a putative two‐component lantibiotic, haloduracin, was identified in the genome of the Gram‐positive bacterium Bacillus halodurans C‐125. We have recently shown that the precursor peptides, HalA1 and HalA2, are processed by the modification enzymes HalM1 and HalM2 in the first in vitro reconstitution of biosynthesis of a two‐component lantibiotic. The activity of each HalM enzyme is substrate specific 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 had occurred. Initial characterization of the dehydration of the peptide substrate HalA2 by HalM2 suggests that this reaction proceeds in a non‐processive manner. The substrate specificity of HalM2 is also examined using truncation mutants of HalA2 that lack all or portions of the leader sequence. Site‐directed mutagenesis of HalM2 is used to identify residues that are important for both dehydration and cyclization.