Manipulation of cyaonobactin biosynthetic enzymes for the production of a diverse range of azol(in)e containing macrocycles on a useful scale, as described for the in
Chapter 1. Introduction 30
vivo and in vitro syntheses, requires a comprehensive understanding of the individual enzymes involved. Much of cyanobactin biosynthesis has been characterised, however some unknowns and uncertainties remain. These studies, through a combination of structural biology, biochemical and biophysical characterisation, aim to address the current unknowns in patellamide biosynthesis, with a focus on using any new insight gained, coupled with our existing knowledge, to develop a biotechnological toolkit for the synthesis of cyanobactins and ultimately diverse cyclic peptides more generally, thus improving upon the existingin vitro synthesis.
There are three specific aspects of the biosynthesis that are investigated in this thesis: (1) Investigation into the role of the C-terminal DUFs - What is their role in nature? Are they involved in synthesis, either directly or indirectly? Do they constitute an essential, or beneficial part of the biotechnological toolkit? (2) Detailed characterisation of the heterocyclase enzyme - What is the mechanism of heterocyclisation? What is the molecular rational governing substrate recognition of the heterocyclase? (3) Investigation into epimerisation - When during synthesis does epimerisation occur? Is it spontaneous or enzymatic? Can the stereochemistry of these residues be controlled? Does an epimerisation step need to be built into the in vitro synthesis?
Finally, these studies aim to develop the in vitro synthesis further, in an attempt to improve the efficiency and flexibility of the process. Ultimately the in vitro synthesis will be tested for its applicability in producing a range of patellamide D analogues in an attempt to determine structural activity relationships for Pgp inhibition.
Chapter 2
Structural and Biochemical
Characterisation of PatG-DUF
2.1
Introduction
The roles of the C-terminal domains of PatA and PatG in patellamide biosynthesis are unknown and hence they are designated as Domains of Unknown Function (DUFs). The high sequence homology between the domains implies their function is related (Fig. 2.1).
Figure 2.1: Sequence alignment of PatA-DUF and PatG-DUFThe conserved C-terminal DUFs of PatA and PatG share 56 % identity. Fully conserved residues are
shaded black and non-conserved residues are unshaded.
Chapter 2. Structural and Biochemical Characterisation of PatG-DUF 32
Moreover both DUFs are highly conserved in all cyanobactin pathways, (some of which can be seen in Figs. 2.2, 2.3), suggesting they have an important role in cyanobactin biosynthesis.
Chapter 2. Structural and Biochemical Characterisation of PatG-DUF 33
Figure 2.2: PatA-DUF homologuesSequence alignment between PatA-DUF and homologues from related cyanobactin pathways. Fully conserved residues are shaded black, partially conserved residues are shaded in grey, non-conserved residues are
Chapter 2. Structural and Biochemical Characterisation of PatG-DUF 34
Figure 2.3: PatG-DUF homologuesSequence alignment between PatG-DUF and homologues from related cyanobactin pathways. Fully conserved residues are shaded black, partially conserved residues are shaded in grey, non-conserved residues are
unshaded.
Broadly speaking, three possibilities for the DUFs exist: (1) In all examples, the C- terminal DUFs are associated with domains exhibiting protease activity. Therefore it is possible the DUFs a↵ect proteolysis - either to accelerate reaction rates (PatApr turnover is very low in isolation[42]), or to regulate activity. (2) PatG is a multi-domain protein, containing an oxidase domain and a macrocyclase domain in addition to the C-terminal domain of unknown function. The oxidase and macrocyclase domains catalyse two, very di↵erent chemical reactions, and both domains are active in isolation[47]. Therefore it is plausible that the DUFs exhibit intrinsic catalysis, chemically distinct from that of their associated domains. In this case, epimerisation of the two residues N-terminal to the thiazole heterocycles is the most likely candidate, as it is the only post-translational modification that hasn’t currently been attributed to a gene product of the patellamide
Chapter 2. Structural and Biochemical Characterisation of PatG-DUF 35
operon. (3) The DUFs might a↵ect patellamide biosynthesis indirectly. They could provide a chaperone-like function, assisting in the pre-organisation of the precursor peptide prior to biochemical modification. Alternatively they might be involved in host- cell immunity, and/or export of the cyclic peptides from the host-cell. It is also possible they are involved in mediating PPIs, providing a sca↵old for multi-enzyme complex formation.
Since one of the on-going goals of the lab is to optimise the established in vitro
patellamide synthesis[47] for the production of a large variety of diverse patellamide- like cyclic peptides on a mg scale, the first two hypothetical roles for the DUFs, as described above, are the most important. As a consequence, the objective of this chapter is to characterise the C-terminal domains of unknown function, focusing on these first two hypothetical roles, to ultimately determinine whether the DUFs would constitute an essential part of a biotechnological ‘toolkit’ for the in vitro synthesis of cyanobactins. We turn to X-ray crystallography in an attempt to gain insight through structural characterisation, and isothermal titration calorimetry (ITC) and nuclear magnetic resonance spectroscopy (NMR) to investigate binding of the DUFs with plausible substrates: the precursor peptide and intermediates formed during biosynthesis. By expressing the DUFs alongside their associated domains, we can investigate what e↵ect if any, the DUFs have on their activity.