Development of a biotechnological toolkit for the synthesis of diverse cyclic peptides
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Date
21/06/2017Author
Supervisor
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Abstract
Cyclic peptides possess desirable characteristics as potential pharmaceutical scaffolds.
The cyanobactin family of cyclic peptide natural products boast diverse structures and
bioactivity. Exemplars are the patellamides, which have attracted attention due to
their ability to reverse the effects of multi-drug resistance in human leukemia cells. In
addition to their macrocyclic architecture patellamides contain azol(in)e heterocycles
and d-amino acids. This structural complexity makes them challenging targets for
chemical synthesis. Understanding their biosynthesis will enable the development
of a biotechnological ‘toolkit’ for the synthesis of new pharmaceutical compounds.
Patellamides are ribosomally-synthesised and post-translationally modified peptides
(RiPPs) and much of their biosynthesis has been elucidated, however there are still
elements of their biosynthesis that are not yet fully understood.
PatA and PatG contain C-terminal domains of unknown function (DUFs). The
crystal structure of PatG-DUF has been solved and subsequent to biochemical and
biophysical investigation PatG-DUF was found not to constitute an essential part of the
biotechnological ‘toolkit’ and can be excluded from in vitro enzyme-based synthesis of
cyanobactin-like cyclic peptides.
The cyanobactin heterocyclases are able to introduce heterocycles into a peptide
backbone, seemingly irrespective of the neighbouring residues; however a molecular
rational governing substrate recognition is unknown. Additionally the mechanism of
heterocyclisaton is disputed. Analysis of crystal structures of LynD in complex with
cofactor and substrate (solved by Dr Jesko Koehnke) enabled the active site and
substrate recognition site to be located. A new mechanism for heterocyclisation has
been proposed. Guided by the substrate recognition observed in complex structures
a constituently active heterocyclase (AcLynD) has been engineered, which is able to
process short, leaderless peptide substrates.
Epimerisation in cyanobactin biosynthesis is believed to be spontaneous, but its precise
timing is uncertain. NMR analysis of selectively labelled peptide substrates processed by
the modifying enzymes, identified epimerisation to be spontaneous on the macrocycle,
regardless of whether the neighbouring heterocycles have been oxidised.
A one-pot in vitro synthesis of cyanobactins has been developed, and employed to create
a number of patellamide D analogues to ascertain structural-activity relationships.
Type
Thesis, PhD Doctor of Philosophy
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