Structural Biochemistry/Lantipeptides

Lantipeptides

Introduction

Lantipeptides were previously known as lantibiotics because of the antimicrobial characteristics they exhibited, but the term has since then been changed to lantipeptides with the discovery of lantibiotics that did not perform antimicrobial tasks. With this discovery, the family that they belong to was extended to over 90 compounds.

Lantipeptides are ribosomally synthesized peptides with thioether cross-links which is formed by dehydration of Serine/Threonine residues and subsequent addition of cysteine residues to the resulting dehydro amino acid meso-lanthionine (Lan) and (2S,3S,6R)-3-methyllanthionine (MeLan. Meso-lanthionine and (2S,3S,6R)-3-methyllanthionine are cross-links which come from posttranslational modification of a precursor peptide.

History

Lantipeptides were previously known as lantibiotics because of the antimicrobial characteristics they exhibited, but the term has since then been changed to lantipeptides with the discovery of lantibiotics that did not perform antimicrobial tasks. With this discovery, the family that they belong to was extended to over 90 compounds.

Lantibiotics have historically been used as antimicrobial agents. One latipeptide (nisin) has been used in the food industry for over 50 years as a preservative. The name lantibiotics was first introduced as an abriation of the term “lanthionine-containing peptide antibiotics” . Work done by Erhard Gross and John L. Morell in the 1960-70’s led to the field of lantibiotics that is around today.

Nisin

The 3D structure of nisin, an antimicrobial used in the food industry.

One well known lantibiotic that has been studied extensively is Nisin. Nisin is composed of 34 amino acids and has been used widely in products such as cheese, meats, and other everyday items as a deterrent against bacteria that can spoil or otherwise infect food.

Recently, studies have been done with nisin that show it may aid in fighting cancer. In a study performed at the University of Michigan, it was found that nisin creates pores in the cell membranes of cancer cells. These pores allow calcium to flow into the cancerous cell, which can ultimately lead to its death. It is unclear how specifically calcium influx can lead to death, but a protein called CHAC1 which is activated by nisin is involved. It was also found that nisin may interrupt the cell cycle of cancer cells, while at the same time leaving regular cells unaffected .

Antimicrobial Characteristics

Mechanism for pore formation in cellular membrane.

One of the most apparent characteristics of lantibiotics is their high activity against bacteria. They are effective against gram positive bacteria such as of Staphylococcus, Streptococcus, Enterococcus, and Clostridium and gram-negative bacteria such as Neisseria. Because of these properties, they have been the subject of much scientific research and have had applications in industries such as food preservation. They have been found to treat various infections, such as Clostridium difficile, and others . Lantipeptides may also have application in other industries, such as agriculture, verterinary medicine, and molecular imaging.

The mechanism through which lantipeptides gain their antimicrobial properties is often through the inhibition of bacterial cell wall synthesis. They are also known to create pores in membranes, which can greatly ruin the integrity of the cell. Specifically, the mechanism through which lantipeptides have been known to act is through the inhibition of transglycosylation. They do this by binding to lipid II, which is an integral part of building the cell membrane .

Mechanism of Nisin

The mechanism through which nisin is able to perform well as an antimicrobial have been well documented, which is one of the reasons it is used extensively today. It binds to lipid II through contacts the A and B rings of pyrophosphate moiety. It then forms pores in the membrane by inserting itself, forming groups of four lipid II molecules and eight nisin peptides.

Classes of Lantipeptides

Lantipeptides can be classified into four distinct classes of biosynthetic enzymes that recognize Lan and MeLan: dehydratase and cyclase enzymes (Class I), bifunctional lanthionine synthetases (Class II), trifunctional synthetases and carbocyclic rings (Class III), trifunctional lanthionine synthetases (Class IV).

In Class I

Class 1 lantipeptides are known as dedicated dehydratase and cyclase enzymes, and for good reason. There are two different enzymes that carry out these processes: dehydratase LanB and cyclase LanC. The LanB genes supply proteins of about 1000 residues that are not homologous to any known enzymes. The LanC genes encode proteins of about 400 residues and present a low sequence identity. Among recently discovered and isolated lantipeptides are a modified peptide from actinomycete of Microbispora corallina, which has high activity against gram-positive bacteria, and planosporicin, which is derived from actinomycete Planomonospora. The NMR structures of these two have shown similarities in conformation.

Class II

Class II lantipeptides are known as bifunctional lanthionine synthetases. They are known to perform dehydration and cyclization reactions. LanM is the bifunctional synthetase that performs these processes. The proteins is produces range from 900-1,2000 residues in length and consist of two domains, which are an N-terminal dehydratase domain that bears no enzyme that is homologous to LanB, and a C-terminal cyclase domain, which a one-fourth sequence identity to LanC, including conservation of the zinc-binding residues essential for NISC catalysis. There have been many new additions to this class of lantipeptide recently. One of these is haloduracin, which is from Bacillus halodurans. This is the first lantipeptide that is from a species that is alkaliphilic, which means it can survive high pH (or alkaline) environments.

Class III

Class III lantipeptides are known as trifunctional synthetases and carbocyclic rings. Biosynthesis are present in the morphogenetic peptide SapB from Streptomyces coelicolor. There is a big difference between class II and class III lantipeptides, which lead to the separate designation. The difference lies in the fact that this peptide does not demonstrate antibiotic activity. Instead, it promotes the growth of vegetative hyphae that is associated with streptomycete sporulation. A putative modifying enzyme known as RamC is contained in the gene cluster. This enzyme resembles the serine/threonine protein kinases and is structured with a C-terminal domain with homologous features that are similar to cyclase domain of LanM. However, the zinc-binding is absent .

In 2010, labyrinopeptins were discovered from actinomycete Actinomadura namibiensis. The trifunctional lantipeptide known as LabKC was found to have homology with the previously known RamC. Labyrinthopeptin, which is one of the modified products of LabKC, was found to help against neuropathic pain in mice. This is a function of lantipeptides previously unobserved .

Class IV

Class IV lantipeptides biosynthesis involves a cryptic gene cluster in Streptomyces venezuelae. The synthestase, VenL, is made up of an N-terminal OspF-like lyase domain with a serine/threonine kinase domain center. Unlike the RamC, the C-terminal cyclase domain of VenL includes the zinc-binding motif that is present in LanC and LanM .

Bioengineering of Lantipeptides

Knowing lantipeptides can help produce large chemical diversity with low genetic cost in a favorable and adaptable strategy. Lantipeptide biosynthesis has been developing by discovering new classes of biosynthetic machinery, new posttranslational modifications, and more lantipeptide-encoding gene clusters. Even though some catalysis mechanism is remain unknown like LanB, however, by discovering methodology of the production in E. coli, helped the investigations of LanB and furthermore, introduction of nonproteinogenic amino acid into lantipeptides by stop-codon suppression technology.

In Vivo Engineering

One way that lantipeptide engineering is performed is in Escherichia coli. This was first performed in 2005, when a truncated nukacin ISK-1 was produced. This was done through the coexpression of nukA and nukM on one vector. In 2011, a modified LanAs was produced. This was done through the coexpression of lanA and lanM. Coexpression of nisA and nisB on a single vector, with nisC on another vector leads to the production of a nisin precursor peptide. This is the only class I lantipeptide produced in E. coli that has been documented .

In Vitro Engineering

The in vitro approach to engineering comes with its own set of problems, mostly due to problems with immunity or export. This approach utilizes synthetic substrates. One drawback to this synthetic method is in proteolysis, which often results in low yields. For this in vitro method to improve, optimization of this step is required .