Ahmed, Hafna
Description
Mycobacterium tuberculosis, the causative agent of Tuberculosis
(TB), is notorious for its ability to persist during infection
and evade the host immune response, although the cellular
mechanisms behind this are not yet fully understood. Like other
mycobacteria, M. tuberculosis produces the cofactor F420 found in
Actinobacteria and Archaea, which is important, through unknown
mechanisms, for its survival during oxidative stress and in the
reactivation of...[Show more] latent infection. This project presents the
characterisation of the largest F420 utilizing protein family in
mycobacteria known as the flavin/deazaflavin dependent
oxidoreductases (FDORs), forming the basis for identifying
potential functional roles of these enzymes that might contribute
to mycobacterial infection and persistence.
While most FDORs in mycobacteria utilise F420 as a cofactor, this
family also includes proteins with other cofactor specificities,
including proteins that utilise FMN, FAD and heme. Five novel
FDOR structures are presented, which, along with previously
available structures, allowed the identification of conserved
motifs to differentiate between FDORs with different cofactor
specificities. Comparisons between the structures also showed
that FDORs have relatively conserved cofactor-binding regions,
while the substrate binding pockets are extensively modified for
functional adaptation.
Their cofactor preference, sequence similarity and structures
allowed the classification of the FDORs into functional groups,
including the previously identified F420H2-dependent quinone
reductases that also activate 4-nitroimidazole pro-drugs approved
for treating multi-drug resistant M. tuberculosis infection. For
the first time, mycobacterial heme oxygenases belonging to this
family were also found, along with novel FAD binding proteins
that could be involved in the hypoxia response that triggers
mycobacterial dormancy. Furthermore, in silico substrate docking
led to the identification of novel F420H2-dependent fatty acid
saturases and F420H2-dependent biliverdin reductases (F-BVRs)
within the FDORs.
Detailed characterisation of the F-BVR Rv2074 from M.
tuberculosis showed that its homologues are present in pathogenic
and commensal mycobacteria and that it reduces biliverdin-IXα
(the principle isomer produced by human macrophages) to
bilirubin-IXα. Bilirubin is a potent antioxidant that could
contribute to M. tuberculosis surviving oxidative stress
encountered inside macrophages. Rv2074 reduces biliverdin by a
mechanism similar to the nicotinamide-dependent reactions in the
mammalian biliverdin reductases as inferred using the structure
of the Rv2074:F420 complex with biliverdin modelled into the
active site. Proton donation to a pyrrole nitrogen occurs first
from a hydroxonium ion stabilised by an arginine residue, which
is consistent with the requirement for an alternate proton donor
since F420H2 appears to be stabilised in its deprotonated state
when bound to FDORs. The resulting cationic intermediate
undergoes hydride transfer with F420H2, completing bilirubin
formation.
Lastly, F420 production was found to be more widespread than in
just Actinobacteria and Archaea, with data confirming F420
production in some Proteobacteria and Chloroflexi, which also
encode the proteins required for its biosynthesis. FDORs are also
present in these organisms, implying their capability of
utilising this rare cofactor as well.
Overall, the work described in this thesis highlights the
diversity of the FDORs and has identified functional roles that
could contribute to M. tuberculosis pathogenesis and persistence
by enhancing survival during oxidative stress.
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