Elucidating and exploiting halogenase recruitment beta hairpin docking domains in nonribosomal peptide biosynthesis

Lead Research Organisation: University of Warwick
Department Name: School of Life Sciences

Abstract

Nonribosomal peptides and polyketides are classes of natural products, comprising a large variety of biologically-relevant compounds. They are synthesised by nonribosomal peptide synthases (NRPSs) and polyketide synthases (PKSs) respectively, which are modular megasynthases that act in an assembly line-like manner. The interaction between subunits of PKS and NRPS proteins is critical to the fidelity of their biosyntheses, and are known in many cases to be facilitated by C-terminal and N-terminal docking domains.
One type of interaction between subunits in hybrid NRPS-PKS systems involves the docking of -hairpin docking domains (HDDs) attached to catalytic domains at the N-terminus of one subunit with a short linear motifs (SLiMs) attached to carrier proteins at the C-terminus of another subunit. Recent computational work in the Challis group has suggested that this system of docking domains may be far more prevalent in the biosynthetic machinery of polyketide and nonribosomal peptide metabolites than first appreciated.
This project will focus upon the characterisation of HDD/SLiM interactions predicted to occur in the biosynthetic pathway of nonribosomal peptides from cyanobacteria. The aims of the project are to experimentally characterise the HDD/SLiM interactions by overproducing the relevant proteins in E. coli and using synthetic substrate analogues to examine their enzymatic activity. We will also attempt to obtain high resolution structural information for the proteins using X-ray crystallography and NMR spectroscopy. Once characterised, this system will be exploited to understand the potential of the HDD/SLiM systems as a tool for engineering of biosynthetic pathways, including the production of hybrid assembly lines using components from the biosyntheses of different metabolites.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M01116X/1 01/10/2015 30/09/2023
1782570 Studentship BB/M01116X/1 03/10/2016 30/09/2020 Matthew John Beech
 
Description The biochemistry of the proteins in the initial steps of aeruginosin biosynthesis have been reconstituted in vitro, confirming the first example of a tailoring enzyme being recruited to a carrier protein by interaction between a beta-hairpin docking domain and a short linear motif. The implications of this are that we could potentially easily tailor the structures of molecules in biosynthetic assembly lines by utilising these dockings domain pairs to direct tailoring enzymes to specific points of the biosynthetic assembly line. This could be used in future research to develop novel compounds with interesting biological activities.

Additionally we have developed and implemented techniques to monitor reactions occuring on molecules appended to carrier proteins and establish their relative kinetics, and work is ongoing to establish the role of docking domains in determining kinetics of these reactions.
Exploitation Route The understanding we are developing of the biophysical and biochemical roles of beta-hairpin docking domains in bacterial biosyntheses will be essential for guiding the ongoing efforts to engineer biosynthetic pathways. This will be essential in the development of new antibiotic, anticancer, immunosuppressant and agrochemical compounds. Therefore, these findings will be of interest to the industrial biotechnology and pharmaceutical sectors.
Sectors Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology