Lantibiotic Reductase LtnJ Substrate Selectivity Assessed with a Collection of Nisin Derivatives as Substrates

Abstract

bLantibiotics are potent antimicrobial peptides characterized by the presence of dehydrated amino acids, dehydroalanine and dehydrobutyrine, and (methyl)lanthionine rings. In addition to these posttranslational modifications, some lantibiotics exhibit additional modifications that usually confer increased biological activity or stability on the peptide. LtnJ is a reductase responsible for the introduction of D-alanine in the lantibiotic lacticin 3147. The conversion of L-serine into D-alanine requires dehydroalanine as the substrate, which is produced in vivo by the dehydration of serine by a lantibiotic dehydratase, i.e., LanB or LanM. In this work, we probe the substrate specificity of LtnJ using a system that combines the nisin modification machinery (dehydratase, cyclase, and transporter) and the stereospecific reductase LtnJ in Lactococcus lactis. We also describe an improvement in the production yield of this system by inserting a putative attenuator from the nisin biosynthesis gene cluster in front of the ltnJ gene. In order to clarify the sequence selectivity of LtnJ, peptides composed of truncated nisin and different mutated C-terminal tails were designed and coexpressed with LtnJ and the nisin biosynthetic machinery. In these tails, serine was flanked by diverse amino acids to determine the influence of the surrounding residues in the reaction. LtnJ successfully hydrogenated peptides when hydrophobic residues (Leu, Ile, Phe, and Ala) were flanking the intermediate dehydroalanine, while those in which dehydroalanine was flanked by one or two polar residues (Ser, Thr, Glu, Lys, and Asn) or Gly were either less prone to be modified by LtnJ or not modified at all. Moreover, our results showed that dehydrobutyrine cannot serve as a substrate for LtnJ. Since the discovery of penicillin by Alexander Fleming in 1928, antibiotics have saved the lives of countless people. Regrettably, due to abuse and overuse, increasing resistance to antibiotics has been found among pathogenic bacteria, which has led to an urgent need for new antimicrobial compounds (1-4). Lanthipeptides, defined as posttranslationally modified peptides containing a lanthionine and/or methyllanthionine ring(s), are a type of ribosomal peptides produced by many Gram-positive bacteria (5, 6). They can be subdivided into 4 different classes based on the enzyme(s) that catalyze(s) the formation of lanthionine residues (6). Some of them (i.e., classes I and II) show antimicrobial activity and are referred to as lantibiotics (7).The capability of lantibiotics to inhibit the growth of obstinate pathogens, including multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, and oxacillin-resistant Gram-positive organisms, makes them very promising candidates for future antimicrobial development (7-9). So far, only a few lantibiotics have been commercially applied or are under development for medical use in spite of their promising properties (7, 10). Nisin, the model class I lantibiotic produced ...

Specificity and Application of the Lantibiotic Protease NisP

“…1% TFA was added to terminate the reaction at 5 different time points (5, 15, 30, 45, and 60 min). All the samples were analyzed by analytical RP-HPLC as indicated before (Mu et al, 2015 ) and measuring the absorbance at 205 nm. For each substrate concentration, the initial velocity was calculated on the basis of the peak area of released nisin vs. time.…”

Mimicry of a Non-ribosomally Produced Antimicrobial, Brevicidine, by Ribosomal Synthesis and Post-translational Modification

“…These increasing achievements will likely make it possible that in the near future D-amino acids can be introduced into various types of RiPPs. Alternatively, some D-amino acids can be introduced into RiPPs by LtnJ-catalyzed hydrogenation of LanB-introduced dehydro-amino acids (Mu et al, 2015).…”

New Insights into the Biosynthetic Logic of Ribosomally Synthesized and Post-translationally Modified Peptide Natural Products

“…Moreover, CrnJ was able to reduce Dha to D-Ala (Lohans et al, 2014). It will be interesting to determine the factors behind this relaxed substrate specificity since most of the reductases acting on dehydroamino acids characterized to date are specific for Dha residues (Cotter et al, 2005, Mu et al, 2015, Yang and van der Donk, 2015). Other RiPPs contain D-amino acids that are introduced via epimerization.…”