Tyrocidine is a mixture of cyclic decapeptides produced by the bacteria ''
Bacillus brevis
''Brevibacillus brevis'' (formerly known as ''Bacillus brevis'') is a Gram-positive, aerobic, motile, spore-forming, rod-shaped bacterium commonly found in soil, air, water, and decaying matter. It is rarely associated with infectious diseases. ...
'' found in soil. It can be composed of 4 different amino acid sequences, giving tyrocidine A–D (See figure 1). Tyrocidine is the major constituent of
tyrothricin
Tyrothricin is an antibiotic mixture which was isolated from '' Bacillus brevis'' by Rene Dubos in the late 1930s. It was later shown by Dubos and Rollin Hotchkiss to be a mixture of two different antibiotics: gramicidin and tyrocidine.
Both ...
, which also contains
gramicidin
Gramicidin, also called gramicidin D, is a mix of ionophoric antibiotics, gramicidin A, B and C, which make up about 80%, 5%, and 15% of the mix, respectively. Each has 2 isoforms, so the mix has 6 different types of gramicidin molecules. They c ...
.
[Pubchem: Tyrocidine and Tyrothricin.] Tyrocidine was the first commercially available antibiotic, but has been found to be toxic toward human blood and reproductive cells. The function of tyrocidine within its host ''B. brevis'' is thought to be regulation of
sporulation
In biology, a spore is a unit of sexual or asexual reproduction that may be adapted for dispersal and for survival, often for extended periods of time, in unfavourable conditions. Spores form part of the life cycles of many plants, algae, f ...
.
Tyrocidines A, B, and C are cyclic decapeptides. The biosynthesis of tyrocidine involves three enzymes. Parts of its sequence are identical to
gramicidin
Gramicidin, also called gramicidin D, is a mix of ionophoric antibiotics, gramicidin A, B and C, which make up about 80%, 5%, and 15% of the mix, respectively. Each has 2 isoforms, so the mix has 6 different types of gramicidin molecules. They c ...
S.
History
In 1939, the American microbiologist
René Dubos
René Jules Dubos (February 20, 1901 – February 20, 1982) was a French-American microbiologist, experimental pathologist, environmentalist, humanist, and winner of the Pulitzer Prize for General Non-Fiction for his book ''So Human An Animal ...
discovered the soil microbe ''Bacillus brevis''. He observed the ability of the microbe to decompose the capsule of
pneumococcus
''Streptococcus pneumoniae'', or pneumococcus, is a Gram-positive, spherical bacteria, alpha-hemolytic (under aerobic conditions) or beta-hemolytic (under anaerobic conditions), aerotolerant anaerobic member of the genus Streptococcus. They are ...
bacterium, rendering it harmless. From the soil microbe ''B. brevis'', he isolated
tyrothricin
Tyrothricin is an antibiotic mixture which was isolated from '' Bacillus brevis'' by Rene Dubos in the late 1930s. It was later shown by Dubos and Rollin Hotchkiss to be a mixture of two different antibiotics: gramicidin and tyrocidine.
Both ...
, which had a high toxicity to a large range of bacteria. Tyrothricin was later found to be a mixture of the peptides gramicidin and tyrocidine. These were observed to have toxic effects in red blood cells and reproductive cells in humans, however, if applied externally as an ointment tyrocidine could also be used as a potent antimicrobial agent.
Dubos's discovery helped revive interest in research on
penicillin
Penicillins (P, PCN or PEN) are a group of β-lactam antibiotics originally obtained from ''Penicillium'' moulds, principally '' P. chrysogenum'' and '' P. rubens''. Most penicillins in clinical use are synthesised by P. chrysogenum using ...
.
Mechanism of action
Tyrocidine has a unique mode of action in which it disrupts the cell membrane function, making it a favorable target for engineering derivatives.
Tyrocidine appears to perturb the lipid bilayer of a microbe's inner membrane by permeating the lipid phase of the membrane. The exact affinity and location of tyrocidine within the phospholipid bilayer is not yet known.
Biosynthesis
The biosynthesis of Tyrocidine is similar to Gramicidin S, and is achieved through the use of
nonribosomal protein synthetases (NRPSs).
Its biosynthesis is via an enzymatic assembly consisting of 3 peptide synthetase proteins, TycA, TycB, and TycC, which contain 10 modules. The different tyrocidine analogues (A–D) are not produced by different enzymes, but rather by an enzyme system that is capable of incorporating different amino acids of structural similarity at specified sites. The amino acid sequence is determined by the organization of the enzyme and not by any RNA template.
The tyrocidine synthetases TycA, TycB, and TycC are encoded on the tyrocidine operon. This consists of the three genes encoding for the three synthetases as well as three additional
open reading frames
In molecular biology, open reading frames (ORFs) are defined as spans of DNA sequence between the start and stop codons. Usually, this is considered within a studied region of a prokaryotic DNA sequence, where only one of the six possible readin ...
(ORFs). These ORFs, labeled as TycD, TycE, and TycF are downstream of the three synthetase genes (see figure 2). TycD &TycE have the highest similarity to members of the
ATP-binding cassette
The ATP-binding cassette transporters (ABC transporters) are a transport system superfamily that is one of the largest and possibly one of the oldest gene families. It is represented in all extant phyla, from prokaryotes to humans. ABC transp ...
(ABC) transporter family which aid in the transport of substrates across a membrane. It has been suggested that the tandem transporters play a role in conferring resistance in the producer cell through tyrocidine secretion. TycF has been identified as a thioesterase (TE) and is similar to other TEs in bacterial operons used for encoding peptide synthetases. However, the precise function of these TEs remains unknown.
The size of the peptide synthetases corresponds to the amount of activation they carry out. TycA is the smallest and activates a single amino acid from one module, TycB is intermediate in size and activates 3 amino acids with 3 modules, and TycC is the largest and activates 6 amino acids with 6 modules (See figure 3).
Each module performs all the catalytic reactions necessary to incorporate a single amino acid onto the peptide chain. This is accomplished through the subdomains for adenylation (A), peptityl carrier protein (PCP), condensation (C), and depending on the amino acid position, an
epimerization
In stereochemistry, an epimer is one of a pair of diastereomers. The two epimers have opposite configuration at only one stereogenic center out of at least two. All other stereogenic centers in the molecules are the same in each. Epimerization is ...
(E). The adenylation subdomain is used in activating the specific amino acid. Each module uses one molecule of the selected substrate amino acid with one molecule of
ATP to give an aminoacyl adenylate enzyme complex and pyrophosphate. The activated amino acid can then be transferred to the enzyme bound 4'-
phosphopantetheine
Phosphopantetheine, also known as 4'-phosphopantetheine, is a prosthetic group of several acyl carrier proteins including the acyl carrier proteins (ACP) of fatty acid synthases, ACPs of polyketide synthases, the peptidyl carrier proteins (PCP), ...
of the carrier protein with the expulsion of
AMP #REDIRECT Amp
{{Redirect category shell, {{R from other capitalisation{{R from ambiguous page ...
from the system. The carrier protein uses the 4'-phosphopantetheine prosthetic group for loading of the growing peptide and their monomer precursors.
Elongation of the peptide chain is achieved through condensation of the upstream PCP onto an adjacent downstream PCP-bound monomer. Some domains have modification subdomains, such as the E subdomain seen in domains 1 and 4 in tyrocidine, which will generate the
D-configured amino acid. On the final module is the TE domain used as a catalyst for cyclization or product release. The release of the product from the carrier protein is achieved through acylation of the active site serine of TE in which the decapeptide is transferred from the thiol ether to the serine residue. Deacylation can then occur through intramolecular cyclization or through hydrolysis to give the cyclic or linear product respectively (See figure 4).
In the case of tyrocidine, ring closure has been shown to be highly favorable due to 4 H-bonds helping the decapeptide backbone to adopt a stable conformation (See figure 5).
This intramolecular cyclization occurs in a head-to-tail fashion involving the N-terminus of the
D-Phe1 and the C-terminus of the
L-Leu10 (See figure 4).
Chemoenzymatic strategies
There is no general biochemical solution for macrocyclization of a peptide chain. Isolated tyrocidine (Tyc) TE domains can be used to cyclize chemically derived peptidyl-thioester substrates, providing a powerful route to new cyclic compounds. In order for this macrocyclization to occur, the peptide chain must be activated at its C-terminus with an ''N''-acetylcysteamine (SNAC)
leaving group In chemistry, a leaving group is defined by the IUPAC as an atom or group of atoms that detaches from the main or residual part of a substrate during a reaction or elementary step of a reaction. However, in common usage, the term is often limited t ...
.
An
alanine scan
In molecular biology, alanine scanning is a site-directed mutagenesis technique used to determine the contribution of a specific residue to the stability or function of a given protein. Alanine is used because of its non-bulky, chemically inert, ...
through the 10 positions of tyrocidine shows that only the
D-Phe and
L-Orn are required for sufficient cyclization.
Tyc TE can also be used biomimetically in which it mimics the environment created by the TE domain with the substrate's PCP through use of a synthetic tether linked to a
polyethylene glycol
Polyethylene glycol (PEG; ) is a polyether compound derived from petroleum with many applications, from industrial manufacturing to medicine. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), depending on its molecular we ...
(PEG) amide resin.
Use of this resin bound to a desired substrate with isolated TE can allow for catalytic release of the resin as well as macrocyclization of the substrate (See figure 6
). Use of
solid phase peptide synthesis
In organic chemistry, peptide synthesis is the production of peptides, compounds where multiple amino acids are linked via amide bonds, also known as peptide bonds. Peptides are chemically synthesized by the condensation reaction of the carboxy ...
(SPPS) allowed the incorporation of a diverse array of monomers into the peptide chain. Later studies used the high tolerance of Tyc TE in order to modify the peptide backbone post-synthetically. This also allowed for glycosylation of the tyrosine or serine residues to be incorporated.
Use of these methods has led to many promising new therapeutic agents.
References
External links
* {{MeshName, Tyrocidine
Polypeptide antibiotics