Synthesising more complex peptides for a specific function

Synthesising more complex peptides for a specific function

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  • 15 September 2023
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Nature not only affords insights into which peptides or proteins might be valuable to produce, but also give insightful clues about how to synthesise them. In nature, there are two main ways that peptides are produced: either utilising the ribosomes or employing a non-ribosomal mechanism. Ribosomal peptide biosynthesis has been studied extensively; each ribosome acts much like a computer, employing a special code to signal and incorporate each amino acid into the overall peptide structure. Non-ribosomal peptide biosynthesis is more complicated, utilising modular multienzyme complexes, called non-ribosomal synthetases. These complexes behave like a factory assembly line, selecting and assembling various building blocks to create the overall structure. These multienzyme complexes can incorporate a much greater range of building blocks than the ribosome and are able to introduce a diverse array of modifications into the peptides they assemble. This makes non-ribosomal peptides more structurally and functionally diverse than their ribosomal counterparts. 

Researchers within our Flagship 3 are studying non-ribosomal peptide assembly lines to reconstitute and re-program them in the laboratory, enabling the creation of complex peptides and proteins that would be otherwise unachievable. To accomplish this, a thorough understanding of the component parts of the assembly lines is essential. One of the most important components is the condensation domain, which forms peptide bonds between successive building blocks incorporated into the peptide chain. It is of critical importance to understand the mechanism used by condensation domains to connect the building blocks. This will enable assembly lines to be reprogrammed to create useful products in the laboratory. 

Along with former-PhD student Candace Ho, our CIs Max Cryle, Greg Challis, and Colin Jackson published a study on the structure of an important condensation domain involved in biosynthesis of the peptide fuscachelin, produced by the thermophlic bacterium Thermobifida fusca. A complex of one of the condensation domains from the assembly line fused to a particular building block was produced by bacterial expression, purified, and crystallised. The crystals were bombarded with high-intensity X-rays at the Australian Synchrotron enabling the three-dimensional structure of the complex to be solved. The structure was compared with those of similar condensation domains from other assembly lines and analysed computationally to identify important features. Further studies were conducted to assess important structural features, including single amino acid mutations, and the role they play in peptide bond formation.  

This work provided key insights into the way condensation domains interact with the building blocks incorporated by non-ribosomal peptide synthetases, paving the way for further studies of these remarkable assembly lines. Additionally, this contributes to ongoing endeavours to develop bespoke assembly lines in the laboratory for sustainable production of diverse peptides with a range of important applications. This project was published in the prestigious journal Nature Communications in May 2021 and has received over 5,300 views, 6 citations, 52 mentions on Twitter, and 5 mentions by news outlets. 

The project showcases a key cross-nodal collaboration between CIs Cryle, Challis, and Jackson at Monash and ANU. It is an important output from our Flagship 3 within the Develop theme.  

 Reference: Izoré, T., Candace Ho, Y.T., Kaczmarski, J.A. et al. 

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