Structural dynamics and ligand binding in kynurenine-3- monooxygenase
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Wilkinson2013.doc (91.78Mb)
Date
29/06/2013Author
Wilkinson, Martin
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Abstract
Kynurenine 3-monooxygenase is a FAD-dependent aromatic hydroxylase (FAH) which is a
widely suggested therapeutic target for controlling the balance of bioactive metabolite
levels produced by the mammalian kynurenine pathway (KP). Prior to starting this work
no structural information was known for the enzyme, with studies of the human form
complicated by the presence of a C-terminal transmembrane helix. The bacterial
Pseudomonas fluorescens enzyme (PfKMO) lacks the transmembrane region and has
been previously characterised by Crozier-Reabe and Moran [1, 2]. Therefore PfKMO,
which shares 32 % sequence identity with the human enzyme, was selected as a target
for structure solution. Initial substrate bound PfKMO crystals showed poor X-ray
diffraction. Subsequent growth optimisation and the generation of a C252S/C461S
PfKMO mutant (dm2) yielded crystals suitable for structure solution. Selenomethioninelabelled
substrate bound dm2 crystals were used to solve the first structure to a
resolution of 3.40 Å. With just one protein molecule per asymmetric unit, a high solvent
content was responsible for the poor diffraction properties of this crystal form. The
overall fold resembled that of other FAH enzymes with a Rossmann-fold based FADbinding
domain above a buried substrate binding pocket. Interestingly PfKMO possesses
an additional, novel C-terminal domain that caps the back of the substrate-binding
pocket on the opposite side to the flavin. Residues proposed to be involved in substrate
binding were identified and shown to be highly conserved among mammalian KMO
sequences.
Subsequently single crystals of substrate-free dm2 PfKMO were obtained and showed
significantly stronger diffraction due to new lattice packing in an orthorhombic space
group bearing four molecules per asymmetric unit. The structure was solved to a
resolution of 2.26 Å and revealed a clear conformational change of the novel C-terminal
domain. This movement opens a potential route of substrate/product exchange between
bulk solvent and the active site. The investigation of a set of C-terminal mutants further
explored the relevance and mechanics of the conformational change. In addition the
presence of chloride ions in the substrate-free crystal growth solution caused a small
number of localised subtle alterations to the structure, with a potential chloride binding
site identified adjacent to the flavin cofactor. This may have relevance for the observed
inhibition of PfKMO activity by monovalent anions – a feature widely common to FAH
enzymes [3].
The first discovered KMO inhibitors were analogues of the substrate L-Kyn, however one
such compound (m-NBA) was recently shown to instigate uncoupled NADPH oxidation
leading to the release of cytotoxic hydrogen peroxide [1]. A set of substrate analogues
were tested and characterised for inhibition of PfKMO. The picture was shown to be
complex as some substrate analogues completely inhibited the enzyme whilst the
binding of some still stimulated low-levels of NADPH oxidation. Crystallographic studies
with m-NBA and 3,4-dichlorobenzoylalanine (3,4-CBA) bound revealed indistinguishable
structures from that of substrate-bound PfKMO. These studies suggest that the analogue
3,4CBA is a potent PfKMO inhibitor whose therapeutic potential may be re-visited. The
previous most potent KMO inhibitor whose structure was not analogous to the substrate
was Ro 61-8048 [4], which unfortunately did not pass pre-clinical safety tests. A novel
series of 1,2,4-oxadiazole amides based on the structure of Ro 61-8048 was created by
Gavin Milne (PhD, University of St Andrews) and tested on PfKMO. Rounds of refinement
led to the discovery and refinement of low nanomolar competitive inhibitors of the
bacterial enzyme. PfKMO was co-crystallised with each of the four most potent
compounds forming a third different lattice arrangement, which yielded structures to
resolutions of 2.15-2.40 Å. The structures displayed conformational changes resembling
the substrate-free fold possibly caused by displacement of a crucial substrate-binding
residue, R84.
Overall the wealth of structural data obtained may be transferable to predictions about
the structural features of human KMO and to the rational design of therapeutic
inhibitors. The potent novel inhibitors tested may additionally present a new exciting
development for the therapeutic inhibition of human KMO.