Posttranslational conversion of L-serines to D-alanines is vital for optimal production and activity of the lantibiotic lacticin 3147

Abstract

As a general rule, ribosomally synthesized polypeptides contain amino acids only in the L-isoform in an order dictated by the coding DNA/RNA. Two of a total of only four examples of L to D conversions in prokaryotic systems occur in posttranslationally modified antimicrobial peptides called lantibiotics. In both examples (lactocin S and lacticin 3147), ribosomally encoded L-serines are enzymatically converted to D-alanines, giving rise to an apparent mistranslation of serine codons to alanine residues. It has been suggested that this conversion results from a two-step reaction initiated by a lantibiotic synthetase converting the gene-encoded L-serine to dehydroalanine (dha). By using lacticin 3147 as a model system, we report the identification of an enzyme, LtnJ, that is responsible for the conversion of dha to D-alanine. Deletion of this enzyme results in the residues remaining as dha intermediates, leading to a dramatic reduction in the antimicrobial activity of the producing strain. The importance of the chirality of the three D-alanines present in lacticin 3147 was confirmed when these residues were systematically substituted by L-alanines. In addition, substitution with L-threonine (ultimately modified to dehydrobutyrine), glycine, or L-valine also resulted in diminished peptide production and/or relative activity, the extent of which depended on the chirality of the newly incorporated amino acid(s).

Fig. 1.

Structure of LtnA1 and LtnA2, with the 23 posttranslationally modified residues shaded gray. The locations of the three d-alanine residues are indicated. Production of and immunity to lacticin 3147 involves 10 genes present on divergent operons. The left-facing operon is responsible for immunity, ltnA1 and ltnA2 are the structural genes, LtnM1 and ltnM2 encode the lantibiotic synthetases required to dehydrate serines and threonines in a sequence-specific manner, and ltnT encodes a dedicated ABC transporter. The gene encoding the LtnJ dehydrogenase, ltnJ, is the final gene in the right-facing operon. The proposed pathway for the conversion of l-serine to d-alanine via dha is also depicted.

Fig. 2.

Bioactivity in an ltnJ knockout. Deletion of ltnJ results in a dramatic loss of activity (no zone in well-diffusion assays) against a sensitive indicator. Partial complementation can be achieved by heterologous expression of ltnJ in trans from pNZ44ltnJ. The loss in activity is primarily caused by reduced production of both peptides as indicated by the HPLC profiles (the profile from pMRC01 and pMRC01ΔltnJ represent 10- and 20-ml eluates, respectively). Deferred antagonism assays confirm that pMRC01ΔltnJ retains some inhibitory capacity observed as a zone of inhibition surrounding a colony. This activity can be enhanced by wild-type LtnA2 but not by LtnA1, confirming that the loss of LtnJ has a relatively greater impact on LtnA2. MS analyses confirmed the loss of 2 and 4 mass units from LtnA1 and LtnA2, respectively, from the ΔltnJ mutant.

Fig. 3.

Masses and HPLC profile of lacticin 3147 mutants (10-fold concentration of eluates). In all instances, the corresponding unmutated sister peptides, where present, had masses corresponding to their wild-type equivalents.