Protonation state of F420H2 in the prodrug-activating deazaflavin dependent nitroreductase (Ddn) from Mycobacterium tuberculosis

The protonation state of the deazaflavin dependent nitroreductase (Ddn) enzyme bound cofactor F420 was investigated using UV-visible spectroscopy and computational simulations. The reduced cofactor F420H2 was determined to be present in its deprotonated state in the holoenzyme form. The mechanistic implications of these findings are discussed.MLC and CJJ gratefully acknowledge funding from the Australian Research Council in the form of Discovery Project funding (DP130102144) and ARC Future Fellowships. MLC also acknowledges generous allocations of supercomputing time on the National Facility of the Australian National Computational Infrastructure

F420: Characterisation and Catalysis in Mycobacterial Enzymes

Experimental UV-visible spectroscopy, computational quantum chemistry and molecular dynamics (MD) simulations have been used to characterise the deazaflavin cofactor F420 and its role in the catalytic activity of a newly classified superfamily of flavin/deazaflavin dependent oxidoreductases (FDORs). Two FDORs were selected to represent the enzyme family as case studies in the detailed investigations; Deazaflavin dependent nitroreductase (Ddn) and Rv2074, which is a novel biliverdin reductase. The main findings of the thesis are as follows. The protonation/deprotonation state of the F420H2 cofactor species was determined in the Ddn active site using experimental UV-visible spectroscopy and computationally simulated spectra. Ddn was found to bind F420H2 in its deprotonated state. This is important as it implies that the subsequent protonation step following the obligate hydride transfer requires a different proton donor for the completion of the reaction. Using the above results and the published crystal structure of Ddn as a starting point, the MD simulations helped reveal the roles the active site residues play in the catalytic mechanism of pretomanid activation. An interesting outcome of this investigation was the role of three non-binding tyrosine residues that formed a hydrophobic barrier, which shielded the hydride transfer site. Hydrophobic shielding of the hydride transfer site by a non-binding tyrosine was also observed in the second case study, Rv2074. As the studies presented in this thesis are the first to use computational modelling on members of the FDOR family, it is unclear whether other members that use F420H2 as a cofactor also demonstrate similar hydrophobic shielding behaviour. Nonetheless, Rv2074 and Ddn belong to different subfamilies in the FDOR superfamily and outside of a couple of conserved residues, they have vastly different active sites with different residues. The conserved residues were found to be involved in binding F420 and hydrophobic shielding. As this feature is found to be conserved in members from different subfamilies, it could provide new insights into the importance of this feature in how F420 dependent reductases catalyse reactions. Finally, high level ab initio calculations were used to determine the reaction pathway of the pretomanid activation in Ddn, the molecular structures of the transition states and intermediates involved in the reaction pathway and their associated relative energy barriers. The initial hydride transfer step was determined to be rate limiting. After hydride transfer the hydrophobic shield is disrupted allowing exposure of the active site to bulk solvent so proton transfer can occur from a hydroxonium ion. The product then undergoes a series of spontaneous intramolecular reactions to form the final reactive nitrogen species. The detailed characterisation of this mechanism could be utilised in the future development of pro-drugs that would undergo similar activation reactions

Hydrophobic Shielding Drives Catalysis of Hydride Transfer in a Family of F420H2-Dependent Enzymes

A family of flavin/deazaflavin-dependent oxidoreductases (FDORs) from mycobacteria has been recently characterized and found to play a variety of catalytic roles, including the activation of prodrugs such as the candidate anti-tuberculosis drug pretomanid (PA-824). However, our understanding of the catalytic mechanism used by these enzymes is relatively limited. To address this, we have used a combination of quantum mechanics and molecular dynamics calculations to study the catalytic mechanism of the activation of pretomanid by the deazaflavin-dependent nitroreductase (Ddn) from Mycobacterium tuberculosis. The preferred pathway involves an initial hydride transfer step from the deprotonated cofactor (i.e., F420H-), with subsequent protonation, before a series of spontaneous intramolecular reactions to form the final reactive nitrogen species. The most likely proton source is a hydroxonium ion within the solvent accessible active site. Intriguingly, catalysis of the rate-determining hydride transfer step is aided by three tyrosine residues that form a hydrophobic barrier around the active site that, upon reaction, is then disrupted to allow increased water accessibility to facilitate the subsequent proton transfer step. The catalytic mechanism we propose is consistent with previous experimental observations of the Ddn enzyme and will inform the design of improved prodrugs in the future.Australian Research Council Grants DP130102144 and DE120102673. M.L.C. and C.J.J. gratefully acknowledge support from the Australian Research Council under their Discovery Projects program, and the award of a DECRA fellowship to C.J.J

Rv2074 is a novel F420H2-dependent biliverdin reductase in Mycobacterium tuberculosis

Bilirubin is a potent antioxidant that is produced from the reduction of the heme degradation product biliverdin. In mammalian cells and Cyanobacteria, NADH/NADPH-dependent biliverdin reductases (BVRs) of the Rossmann-fold have been shown to catalyze this reaction. Here, we describe the characterization of Rv2074 from Mycobacterium tuberculosis, which belongs to a structurally and mechanistically distinct family of F420H2-dependent BVRs (F-BVRs) that are exclusively found in Actinobacteria. We have solved the crystal structure of Rv2074 bound to its cofactor, F-420, and used this alongside molecular dynamics simulations, site-directed mutagenesis and NMR spectroscopy to elucidate its catalytic mechanism. The production of bilirubin by Rv2074 could exploit the anti-oxidative properties of bilirubin and contribute to the range of immuno-evasive mechanisms that have evolved in M. tuberculosis to allow persistent infection

Sequence-Structure-Function Classification of a Catalytically Diverse Oxidoreductase Superfamily in Mycobacteria

The deazaflavin cofactor F420 enhances the persistence of mycobacteria during hypoxia, oxidative stress, and antibiotic treatment. However, the identities and functions of the mycobacterial enzymes that utilize F420 under these conditions have yet to be resolved. In this work, we used sequence similarity networks to analyze the distribution of the largest F420-dependent protein family in mycobacteria. We show that these enzymes are part of a larger split β-barrel enzyme superfamily (flavin/deazaflavin oxidoreductases, FDORs) that include previously characterized pyridoxamine/pyridoxine-5'-phosphate oxidases and heme oxygenases. We show that these proteins variously utilize F420, flavin mononucleotide, flavin adenine dinucleotide, and heme cofactors. Functional annotation using phylogenetic, structural, and spectroscopic methods revealed their involvement in heme degradation, biliverdin reduction, fatty acid modification, and quinone reduction. Four novel crystal structures show that plasticity in substrate binding pockets and modifications to cofactor binding motifs enabled FDORs to carry out a variety of functions. This systematic classification and analysis provides a framework for further functional analysis of the roles of FDORs in mycobacterial pathogenesis and persistence.This work was supported by Australian Research Council research grants (DE120102673 and DP130102144) awarded to C.J.J., Commonwealth Scientific and Industrial Research Organisation Office of the Chief Executive Postdoctoral Fellowship awarded to C.G., and Australian National University Higher Degree by Research PhD scholarships awarded to F.H.A., B.M.L., and A.E.M. C.J.J. and P.D.C. thank the Australian Synchrotron for beamtime

Physiology, Biochemistry, and Applications of F420- and Fo-Dependent Redox Reactions

5-Deazaflavin cofactors enhance the metabolic flexibility of microorganisms by catalyzing a wide range of challenging enzymatic redox reactions. While structurally similar to riboflavin, 5-deazaflavins have distinctive and biologically useful electrochemical and photochemical properties as a result of the substitution of N-5 of the isoalloxazine ring for a carbon. 8-Hydroxy-5-deazaflavin (Fo) appears to be used for a single function: as a light-harvesting chromophore for DNA photolyases across the three domains of life. In contrast, its oligoglutamyl derivative F420 is a taxonomically restricted but functionally versatile cofactor that facilitates many low-potential two-electron redox reactions. It serves as an essential catabolic cofactor in methanogenic, sulfate-reducing, and likely methanotrophic archaea. It also transforms a wide range of exogenous substrates and endogenous metabolites in aerobic actinobacteria, for example mycobacteria and streptomycetes. In this review, we discuss the physiological roles of F420 in microorganisms and the biochemistry of the various oxidoreductases that mediate these roles. Particular focus is placed on the central roles of F420 in methanogenic archaea in processes such as substrate oxidation, C1 pathways, respiration, and oxygen detoxification. We also describe how two F420-dependent oxidoreductase superfamilies mediate many environmentally and medically important reactions in bacteria, including biosynthesis of tetracycline and pyrrolobenzodiazepine antibiotics by streptomycetes, activation of the prodrugs pretomanid and delamanid by Mycobacterium tuberculosis, and degradation of environmental contaminants such as picrate, aflatoxin, and malachite green. The biosynthesis pathways of Fo and F420 are also detailed. We conclude by considering opportunities to exploit deazaflavin-dependent processes in tuberculosis treatment, methane mitigation, bioremediation, and industrial biocatalysis

Hydrophobic Shielding Drives Catalysis of Hydride Transfer in a Family of F₄₂₀H₂‑Dependent Enzymes

A family of flavin/deazaflavin-dependent oxidoreductases (FDORs) from mycobacteria has been recently characterized and found to play a variety of catalytic roles, including the activation of prodrugs such as the candidate anti-tuberculosis drug pretomanid (PA-824). However, our understanding of the catalytic mechanism used by these enzymes is relatively limited. To address this, we have used a combination of quantum mechanics and molecular dynamics calculations to study the catalytic mechanism of the activation of pretomanid by the deazaflavin-dependent nitroreductase (Ddn) from <i>Mycobacterium tuberculosis</i>. The preferred pathway involves an initial hydride transfer step from the deprotonated cofactor (i.e., F₄₂₀H^–), with subsequent protonation, before a series of spontaneous intramolecular reactions to form the final reactive nitrogen species. The most likely proton source is a hydroxonium ion within the solvent accessible active site. Intriguingly, catalysis of the rate-determining hydride transfer step is aided by three tyrosine residues that form a hydrophobic barrier around the active site that, upon reaction, is then disrupted to allow increased water accessibility to facilitate the subsequent proton transfer step. The catalytic mechanism we propose is consistent with previous experimental observations of the Ddn enzyme and will inform the design of improved prodrugs in the future

Synthetically controlling dendrimer flexibility improves delivery of large plasmid DNA

Tools for editing the genome and epigenome have revolutionised the field of molecular biology and represent a new frontier in targeted therapeutic intervention. Although efficiencies and specificities of genome editing technologies have improved with the development of TALEs and CRISPR platforms, intracellular delivery of these larger constructs still remains a challenge using existing delivery agents. Viral vectors, including lentiviruses and adeno-associated viruses, as well as some non-viral strategies, such as cationic polymers and liposomes, are limited by packaging capacity, poor delivery, toxicity, and immunogenicity. We report a highly controlled synthetic strategy to engineer a flexible dendritic polymer using click chemistry to overcome the aforementioned delivery challenges associated with genome engineering technologies. Using a systematic approach, we demonstrate that high transfection efficiencies and packaging capacity can be achieved using this non-viral delivery methodology to deliver zinc fingers, TALEs and CRISPR/dCas9 platforms.This work was funded by the Australian Research Council (ARC) and the National Health & Medical Research Council (NHMRC) of Australia

Predicting nitroimidazole antibiotic resistance mutations in Mycobacterium tuberculosis with protein engineering

Our inability to predict which mutations could result in antibiotic resistance has made it difficult to rapidly identify the emergence of resistance, identify pre-existing resistant populations, and manage our use of antibiotics to effectively treat patients and prevent or slow the spread of resistance. Here we investigated the potential for resistance against the new antitubercular nitroimidazole prodrugs pretomanid and delamanid to emerge in Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). Deazaflavin-dependent nitroreductase (Ddn) is the only identified enzyme within M. tuberculosis that activates these prodrugs, via an F420H2-dependent reaction. We show that the native menaquinone-reductase activity of Ddn is essential for emergence from hypoxia, which suggests that for resistance to spread and pose a threat to human health, the native activity of Ddn must be at least partially retained. We tested 75 unique mutations, including all known sequence polymorphisms identified among ~15,000 sequenced M. tuberculosis genomes. Several mutations abolished pretomanid and delamanid activation in vitro, without causing complete loss of the native activity. We confirmed that a transmissible M. tuberculosis isolate from the hypervirulent Beijing family already possesses one such mutation and is resistant to pretomanid, before being exposed to the drug. Notably, delamanid was still effective against this strain, which is consistent with structural analysis that indicates delamanid and pretomanid bind to Ddn differently. We suggest that the mutations identified in this work be monitored for informed use of delamanid and pretomanid treatment and to slow the emergence of resistance