Pacidamycin: an unusual antibiotic with a curious biogenesis. Unravelling the intriguing construction of the peptide backbone.

The majority of medicines in the clinic are based on natural products with a considerable proportion of these being assembled by multifunctional enzyme assembly lines; this can be paralleled with automobile assembly lines with different sections of the enzyme responsible for introducing each component of the natural product. Pacidamycin is an antibiotic with a clinically unexploited mode of action and good selective activity against Pseudomonas aeruginosa, a problem pathogen for burns patients and cystic fibrosis sufferers in particular. We have demonstrated that this structurally unusual natural product is generated by an intriguing nonribosomal peptide synthetase assembly line. Investigations into the construction of this structurally unusual antibiotic are likely to provide fundamental insights as to how different sections of multifunctional enzymatic assembly lines communicate and interact. Understanding the molecular machinery will enable us to harness this and other antibiotic factories in order to enable expeditious access to new designer natural products. Our investigations will also inform on the assembly of two unusual amino acids that are components of pacidamycin. Diaminobutyric acid is a component of a number of biologically important natural products, and whilst sixteen chemical syntheses of this compound have been reported, little is known about its natural assembly. We will determine whether the biogenesis might be tamed in order to enable a biological one-step synthesis of this amino acid and its analogues. The meta-tyrosine residue is also highly unusual and we will carry out investigations into its assembly.

There is an urgent need for new antibiotics with novel architectures. Though resistance to existing antibiotics is increasing at an alarming rate, only 4 new structural classes of antibiotics have been introduced to the clinic in the last 50 years. The antimicrobial nucleoside antibiotics that target bacterial cell wall assembly have attracted much recent attention; these compounds demonstrate good and selective antibacterial activity. A sub-class, the uridyl peptide antibiotics (members include pacidamycin, sansanmycin, mureidomycin and napsamycin) exhibit a clinically unexploited mode of action and numerous biosynthetically unique and intriguing features. We have recently identified the first gene cluster encoding the biosynthesis of a uridyl peptide antibiotic and our heterologous expression studies have enabled the generation of pacidamycins within an alternative host system. The unique structure of the pacidamycins, which includes a pseudo-tetrapeptide backbone incorporating two inversions in the sense of the peptide and two nonproteinogenic amino acids (diaminobutyric acid and meta-tyrosine), render this a curious and potentially highly rewarding system for study. The biosynthetic gene cluster is concise and contains both modular NRPSs and discrete NRPS components. The cluster will provide a useful model system for gaining insight into the fundamentals of the biosynthesis of nonlinear nonribosomal peptides - an area that so far has had little investigation. Our studies will involve complementary approaches of genetic manipulation, in vitro biochemical characterisation of proteins, and feeding studies with synthetic precursors.

1) The proposed research is likely to be of benefit to Biotech such as Biotica, Novacta, Aquapharm, Cubist. Large pharmaceutical companies such as GSK, AZ and Pfizer may benefit from the development of a 1-step synthesis of enantiomerically pure diamino acids. These companies all share an interest in the development of anti-infectives. Findings deemed to be of potential commercial significance and of benefit to UK Plc will be patented prior to dissemination. UEA has a dedicated staff with expertise in evaluating commercialisation prospects and engaging with business. 2) The research will be of interest to the wider biosynthetic and medicinal chemistry communities. The majority of antibiotics in the clinic are based on natural products and many of these are generated by modular, multifunctional enzymes such as polyketide synthases and nonribosomal peptide synthetases. A greater fundamental understanding as to the way in which individual modules communicate and function will enable informed reprogramming of biosynthetic pathways. 3) The PDRA as well as other members of the Goss group will benefit from the project. The PDRA will be involved in tailor-made training programmes that UEA runs, with specific workshops in scientific communication, management, and career development. 4) The general public will benefit from outreach events and the dissemination of the research findings to the wider community. Goss has been involved in numerous demonstration lectures with secondary school children. It is planned that the Goss group will present research findings through a public lecture. Goss and the PDRA will apply to present aspects of the research at a Royal Society Summer Exhibition. 5) In the long term, the research may be of direct or indirect benefit to public health.