Team:Queens Canada/Background

Team:Queens Canada

Non-Ribosomal Peptide Synthetases

The current drug market is dominated by drugs of biosynthetic origin. Greater than 70% of these essential compounds are commonly found in the bacterial kingdom and a significant portion are produced by nonribosomal peptide synthetases (NRPSs). NRPSs are large, multi-modular enzymes that create a variety of structurally and functionally diverse, biologically-active peptides, appropriately named nonribosomal peptides (NRPs). These short peptides often have unique modifications that aid in determining their biological activity, such as heterocyclizations, halogenations, and epimerizations.

Figure 2. (Shown left) Nonribosomal peptide synthetases contain multiple modules, with each module containing several catalytic domains (shown in different colours) each responsible for a specific function.

These large synthetases are composed of a string of modules where each module is responsible for the addition of one amino acid to the growing peptide chain, similar to the function of transfer RNA (tRNA) and codons in ribosomal peptide synthesis. Each module is composed of multiple catalytic domains, each carrying out a specific function during the addition of a new amino acid. The condensation (C) domain is responsible for coupling the new amino acid to the previous amino acid of the peptide chain via dehydration reaction, forming a new peptide bond. The adenylation (A) domain activates new amino acids to be added using ATP. Both the C and A domains contain residues that confer specificity to the amino acid being added by the module. The thiolation/peptidyl carrier protein (T or PCP) domain propagates the growing peptide chain using a mobile thiol arm, which swings to transfer the peptide chain from one module to the next. The final domain of the last module in each NRPS system is usually the thioesterase (TE) domain, which facilitates the release of the final peptide product through hydrolysis or macrocyclization, resulting in a free linear or cyclized peptide chain.

Figure 3. Nonribosomal peptide synthetases are composed of multiple modules, and within each module can be found 3 essential domains: the condensation (C), adenylation (A), and thiolation (T) domains which all work together to add one amino acid to the growing peptide chain.

NRPS systems are a source of antibacterial, antifungal, and anticancer compounds that are important to the pharmaceutical industry. Examples of NRPS-synthesized compounds include cyclosporine A (an immunosuppressant) and vancomycin (an antibacterial compound). These amazing cellular machines produce only one major product through efficient enzymatic stereospecific and regiospecific steps, and these products are usually cyclic (conferring greater stability). However, NRPS-synthesized compounds are also difficult to synthesize through total chemical synthesis to upscale production, which means that optimizing drug leads may be difficult. Nonetheless, NRPS still displays vast potential for novel drug biosynthesis.

Figure 4. Vancomycin (left) and cyclosporine A (right), two therapeutic compounds that are NRPS-synthesized and both contain cyclic structural components (a common characteristic of NRPS products).

There are a number of applications to this year's project. Queen's iGEM's research aims to pave future studies of NRPS systems and NRPs by laying down a foundation of modifying NRPS systems and tagging NRPs for quantification. The long term goal of this research would be to rationally design and produce libraries of NRP drugs with minimized toxicity and realistic ADME (absorption, distribution, metabolism, excretion) properties that could then be tested for biological activity. To extend from this, useful but toxic NRPs could be modified through the addition or removal of specific funtional groups in an attempt to maintain biological activity while also reducing toxicity. Tyrocidine (shown right), for example, is another antibiotic nonribosomal peptide but, unlike vancomycin or cyclosporine, is toxic to the human body. Such NRPs could find a niche use in a therapeutic or industrial setting. Furthermore, the study of NRPS systems could give the scientific community insight into the evolution of NRPS systems in bacteria and fungi and perhaps use it as another measure of relatedness among species or strains of the same species.