Structural Biochemistry/Cell Organelles/Ribosome/RNP

Overview
Ribosomally synthesized natural products (RNPs) are a class of peptides that are of interest to scientists because of their great diversity courtesy of post-translational modifications (PTMs) like hetero- or macrocy-clization, dehydration, acylation, glycosylation, halogenation, prenylation, and epimerization. These RNPs have great potential because PTMs with respect to structure and biological activity compared to the traditional 20 amino acids. They are also favorable because RNPs have relatively short biosynthetic pathways and like to interact with numerous other compounds.

Lantipeptides
A group among RNPs that is of particular interest is the lantipeptides. They are polycyclic peptides that contain the thioether cross-linked amino acids meso-lathionine (Lan) and (2S,3S,6R)-3-methyllanthionine (MeLan). Lantipeptides are all synthesized on the ribosome as a precursor LanA peptide, which contains both a leader peptide, the N-terminal portion of the precursor peptide important for production, but unmodified and cleaved from the final product and a core peptide, the C-terminal portion of the precursor peptide that becomes the final product after modification and leader peptide removal. There are four classes of lantipeptides: class I, class II, class III, and class IV. Class I lantipeptides perform serine/threonine dehydration and thioether cyclization by the dehydratase LanB and the cyclase LanC, respectively. Class II lantipeptides have LanM, a single bifunctional lantipeptide synthetase containing N-terminal dehydratase and C-terminal LanC-like cyclase domains. Class III lantipeptides are modified by a trifunctional synthetase with an N-terminal lyase domain, a central kinase domain, and a putative C-terminal cyclase domain. Finally, class IV lantipeptides have LanL, which contains N-terminal lyase and kinase domains as in class III, but its C-terminal cyclase domain is analogous to LanC.

Lantipeptides were originally thought to be only produced by a specific group of gram-positive Firmicutes, but genomic analysis has shown that other organisms also produce lantipeptides as well like actinomycetes, bacteroidetes, and chlamydiae. A recent study identified lantipeptide biosynthetic genes in 478 of 1,466 bacterial genomes. This shows how abundant lantipeptides are in biological organisms and can be found and harvested from a pool of organisms for researchers to use to do experiment involving these products.

Lantipeptides are interesting to scientists because they have antimicrobial properties, but researchers are finding numerous other uses for them as well. For example, there is interest in using lantipeptides as chemotherapeutics. This idea came from the use of the lantipeptide nisin as a food preservative for more than 50 years because of its use has not presented significant microbial resistance.

Lantipeptides also have a lot of potential in the pharmaceutical industry. They have been proven effective against gram-positive bacteria, including drug-resistant strains like Staphylococcus, Streptococcus, Enterococcus, and Clostridium, and certain gram-negative pathogens like Neisseria and Helicobacter. A few drugs made from lantipeptides are duramycin for the treatment of cystic fibrosis and a derivative of actagardine for the treatment of Clostridium difficle Mutacin 1140 is currently in the developmental stages for the treatment of gram-positive bacterial infections. Other than pharmaceuticals, lantipeptides have applications in agriculture, veterinary medicine, and molecular imaging, proving their diversity in biochemical activity.

Researchers have studied the mechanisms by which lantipeptides conduct biological activity and it has been observed that most antibacterial lantipeptides inhibit cell wall biosynthesis and/or disrupt membrane integrity through pore formation. Lantibiotics, lantipeptides with antimicrobial activity, target and bind to the essential cell wall precursor lipid II. This inhibits the reaction required to synthesize peptiodglycan, an essential part of the cell membrane.

The unique thioether cross-links of lantipeptides are added on post-translationally by biosynthetic enzymes via dehydration of serine or threonine to the α,β-unsaturated residues 2,3-didehydroalanine (Dha) or (Z)-2,3-didehydrobutyrine (Dhb), respectively, followed by Michael-type addition of a cysteinyl thiol to give a thioether bridge. The cross-links are essential for the activity of the compound and for maintaining stability against proteolysis and heat denaturation. This makes lantipeptides quite durable in biological conditions that would otherwise denature other peptides.

Class I
In order to synthesize class I lantipeptides, two separate enzymes carry out reactions forming thioether cross-links: dehydratase LanB and the cyclase LanC. LanB genes encode proteins of about 1,000 residues, while the lanC genes encode proteins of approximately 400 residues. It is believed that these reactions involve a zinc ion bound by a cysteine-cysteine-histidine triad at an active site.

In Vivo Engineering
Currently techniques are being developed to improve the pharmacological properties of lantipeptides, including potency, stability, and solubility. One approach is in vivo lantipeptide production in E. coli. This simplifies genetic manipulation and cuts costs because E. coli is relatively easy to grow and is a well-studied organism. A 2011 study demonstrated the versatility of E. coli production of modified LanAs. This study also represents the only report to date of class I lantipeptide production in E. coli. Two other E. coli expression systems reported in 2011 have utilized dedicated lantipeptide proteases to produce mature lantipeptides. One system produced a fourfold improvement in yield compared to the producing strain. Using E. coli appears to be a viable in vivo method for producing lantipeptides and further research will likely yield more effective methods.

Chemical Synthesis
In addition to using biological techniques to acquire lantipeptides, chemical methods have shown some promise as well. Before 2008 the only successful total chemical synthesis of a lantipeptide was the impressive solution-phase synthesis of nisin. However, recently the first first solid-supported total synthesis of a lantipeptide containing overlapping cross-links, the α-peptide of lacticin 3147, has been reported. This expands the possibilities for synthesizing lantipeptides and should be studied further in conjunction with bioengineering techniques.