P450-catalysed glycopeptide biosynthesis
Specific cytochrome P450 enzymes are essential for the biosynthesis of glycopeptide antibiotics such as vancomycin that are used as a last resort for the treatment of life-threatening bacterial infections. However, an increasing number of bacteria are developing resistances to these antibiotics as well. Researchers from the Max Planck Institute in Heidelberg are investigating the structure and function of these enzyme-antibiotics complexes in order to find ways to use P450s as biocatalysts for the semi-synthetic synthesis of glycopeptide antibiotic analogues that are effective against antibiotic-resistant bacteria.
Vancomycin and teicoplanin are glycopeptide antibiotics that are used as a last resort treatment for a number of life-threatening bacterial infections when standard antibiotics fail due to bacterial resistance or allergies. They were originally discovered in soil-dwelling Streptomyces bacteria. Vancomycin was discovered in 1953, isolated from a bacterial species found in a soil sample collected by a missionary in Borneo and sent to scientists at Eli Lilly and Company.
Vancomycin and teicoplanin consist of seven amino acids, some of which are modified before being joined to a heptapeptide chain and then further modified by the addition of glycosyl groups and the oxidative cross-linking of the side chains (e.g. by the phenolic coupling of aromatic residues.) The linear molecule then assumes the complex three-dimensional ring structure needed for the peptides to act as antibiotics. The biosynthesis of vancomycin and teicoplanin differs from that of larger peptide hormones that are biosynthesised at the ribosomes in that they are synthesised by multifunctional protein complexes called non-ribosomal peptide synthetases (NRPS). The glycopeptide antibiotics remain bound to one of the components (i.e. the peptidyl carrier protein) of the non-ribosomal peptide synthetase machinery as the peptide is synthesised.
A huge variety of CYPs
Dr. Max Cryle, head of a group of researchers in the Department of Biomolecular Mechanisms at the Max Planck Institute for Medical Research (MPImF) in Heidelberg, explores the biosynthesis of glycopeptide antibiotics with the aim of developing new antibiotics analogues that are effective against multidrug-resistant pathogenic bacteria. He is specifically interested in the phenolic coupling of aromatic residues that gives the antibiotics their three-dimensional structure. These cross-links generate the three-dimensional structure needed for these heptapeptides to act as antibiotics. The formation of these phenolic cross-links has been shown to be catalysed by three to four enzymes belonging to the cytochrome P450 (CYP) protein superfamily.
CYPs are enzymes that catalyse the scission of the bond between dioxygen molecules. They are able to transfer one oxygen atom to a broad range of different substrates while the other oxygen atom is reduced to water. This reaction is referred to as a monooxygenase reaction and takes place at the iron atom of the heme complex embedded in the polypeptide chain of the CYP protein. Differences in the protein scaffold lead to variations in substrate specificity and oxidation reaction. The number 450 in the name cytochrome P450 reflects the sharp band at a wavelength of 450, which is the enzyme’s absorption maximum when carbon monoxide forms complexes with the central iron atom. There are few protein families as large and diverse as the cytochrome P450 superfamily, both in terms of the reactions catalysed and the enzymes’ biological functions. The University of Tennessee collects information about P450 genes and currently lists more than 21,000 different CYP genes in all forms of life, including microorganisms, fungi, plants and animals, with 57 different CYP genes in humans (The Cytochrome P450 Homepage - P450 Stats, August 2013).
Modes of action and development of resistance
The CYPs involved in the biosynthesis of glycosylated heptapeptide antibiotics catalyse oxidative reactions such as the phenolic coupling and require the emerging peptide antibiotic to remain bound to the carrier protein of the NRPS complex by way of a sulphide bridge. They exhibit either limited or no activity with free peptide substrates.
Cryle and his team of researchers have studied the structures and biochemical characteristics of the CYP-carrier protein complexes in detail in order to understand the role of each CYP in the biosynthesis of glycopeptide antibiotics. The researchers rely on their own extensive experience as well as the know-how at the MPImF, one of the world’s leading institutions in the field of structural biology of protein complexes and their interactions. The researchers from Heidelberg have already deciphered the three-dimensional structure of OxyE, a CYP enzyme involved in the biosynthesis of teicoplanin. They have also been instrumental in the characterisation of the antibiotics’ catalytic reaction capacity. The researchers have found that the active centre of OxyE differs from the active centres of other CYP enzymes involved in the reaction.
The structural analysis provides an explanation for the antibiotic effect of vancomycin and related glycopeptides and for the formation of vancomycin resistance in some bacterial strains. These antibiotics remain effective against Gram-positive bacteria such as staphylococci, streptococci, enterococci and clostridia by inhibiting the biosynthesis of the bacterial cell wall. They bind to bacterial cell wall precursor peptides and prevent their cross-linking. These peptidoglycans (referred to as PG unit in the schematic) terminate normally in two D-alanine residues (D-Ala-D-Ala) that form five relatively strong hydrogen bonds with vancomycin. In vancomycin-resistant bacteria, the terminal D-alanine is replaced by D-lactate, which results in the loss of a binding site for a hydrogen bond. The antibiotic’s ability to bind to the peptidoglycan is drastically reduced and the cell wall precursor molecules can then form stable cross-links.
New antibiotics against vancomycin-resistant bacteria
Cryle's team is currently working on the development of a semi-synthetic process for making glycopeptide antibiotics. This involves the chemical synthesis of the required peptide precursors and the enzymatic conversion into the mature, biologically active antibiotic through specific, characterised CYPs. This approach has the potential to produce novel glycopeptide molecules that could be tested for their effect against resistant bacterial strains, something that is difficult to achieve with classical organic synthesis methods.
Since the discovery of the first vancomycin-resistant enterococci in 1987, a growing number of bacteria is becoming resistant to vancomycin and teicoplanin. This alarming development is most likely due to the overuse and inappropriate use of antibiotics (see BIOPRO article entitled “Call for responsible antibiotics prescription”). Another reason for the growing number of vancomycin-resistant bacteria might be avoparcin, a glycopeptide antibiotic that is closely related to vancomycin and teicoplanin. Up until 1996, avoparcin was used in agriculture in Germany and elsewhere as an additive to livestock feed to promote the growth of chickens and other animals and to prevent diseases in poultry. The application of avoparcin is now forbidden throughout the EU.
Hospitals and doctors urgently require new chemotherapeutic drugs that are effective against multidrug-resistant bacteria. Precise knowledge of the biosynthesis and the possible interventions targeting specific synthesis steps and enzymes could be used for the development of 3rd generation antibiotics which would help us to regain the upper hand in the constant fight against newly evolving drug-resistant pathogens.