Structure, function and evolution of flavin/deazaflavin dependent oxidoreductases (FDORs) in mycobacteria

Abstract

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 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.