Functions of nitroreductases in mycobacterial physiology and drug susceptibility

Abstract

Tuberculosis is a respiratory infection that is caused by members of the Mycobacterium tuberculosis complex, with M. tuberculosis (Mtb) being the predominant cause of the disease in humans. The approval of pretomanid and delamanid, two nitroimidazole-based compounds, for the treatment of tuberculosis encourages the development of more nitro-containing drugs that target Mtb. Similar to the nitroimidazoles, many antimycobacterial nitro-containing scaffolds are prodrugs that require reductive activation into metabolites that inhibit the growth of the pathogen. This reductive activation is mediated by mycobacterial nitroreductases, leading to the hypothesis that these nitroreductases contribute to the specificity of the nitro prodrugs for mycobacteria. In addition to their prodrug-activating activities, these nitroreductases have different native activities that support the growth of the bacteria. This review summarizes the activities of different mycobacterial nitroreductases with respect to their activation of different nitro prodrugs and highlights their physiological functions in the bacteria.

Keywords: Mycobacterium; antimicrobials; nitroreductase.

Fig 1

The conservation of mycobacterial nitroreductases across different species. The color gradient is the percentage homology of the amino acid sequences of the enzymes with respect to the M. tuberculosis H37Rv homolog (for Rv2032, Rv2466c, Rv3131, Rv3368c, Rv3547, and Rv3790) or M. smegmatis MC²-155 homolog (for MSMEG_6504). Homologs were determined using a threshold e-value of 1 × 10⁻¹⁴. The color shade for each species represents the type of infection caused, yellow/tuberculosis, gray/leprosy, and green/nontuberculous mycobacterial infection.

Fig 2

Schematic on the native activity of Ddn. First, FbiA, FbiB, FbiC, and FbiD work together in the biosynthesis of cofactor F₄₂₀. This oxidized cofactor can be converted into the reduced form through the activity of Fgd in the pentose phosphate pathway. The reduced cofactor can be used by Ddn to reduce menaquinone into the menaquinol form in a two-electron transfer. Menaquinol can subsequently transfer electrons to the terminal cytochrome bd or bc₁-aa₃ oxidases, leading to the reduction of oxygen and energy production. Alternatively, in the absence of the menaquinone-reductase activity of Ddn, menaquinone is reduced in a one-electron transfer to unstable semiquinone that can react with molecular oxygen to form superoxide radicals, leading to the death of the cells.

Fig 3

Schematic on the activation of different F₄₂₀-dependent compounds. The reduced cofactor F₄₂₀, that is produced by Fgd, is used by nitroreductases to reductively activate different nitro-containing compounds. Ddn is the exclusive nitroreductase for pretomanid and delamanid. HC2209, HC2210, HC2211, and JSF-2019 depend on Ddn and another F₄₂₀-dependent nitroreductase that is unknown. CGI-17341, Lee-366, Lee-490, and Lee-562 need the reduced cofactor F₄₂₀ for their activation, although the utilizing nitroreductase is yet to be discovered. Upon reductive activation, nitric oxide species are proposed to be produced, and these inhibit the biosynthesis of mycolic acid and poison the electron transport chain, leading to growth inhibition and death.

Fig 4

Activity of DprE1 mechanism-based inhibitors and DprE2 inhibitors. DPR (decaprenylphosphoryl-β-D-ribose) is converted to DPX (decaprenylphosphoryl-D-2-keto-ribose) through the catalytic activity of DprE1. This reaction produces the reduced form of the bound coenzyme, FADH₂, and the oxidized coenzyme is regenerated through the oxidizing activities of menaquinol or molecular oxygen. However, nitro-containing DprE1 inhibitors can regenerate the bound oxidized coenzyme through a DprE1-mediated activity, forming a nitroso intermediate of the inhibitors. Subsequently, the nitroso intermediates form a covalent bond with the thiol group of Cys384 at the active site of DprE1. This covalent modification inhibits the activity of DprE1. The DPX, that is produced by DprE1, is converted to DPA through the activity of DrpE2. DPA is the sole source of arabinosyl groups that are used in the biosynthesis of the lipoarabinomannan and arabinogalactan components of the mycobacterial cell envelope. Pretomanid and delamanid inhibit the activity of DprE2 by forming an adduct with the protein.

Fig 5

Activation mechanism of Rv2466c-dependent compounds. Two cysteine residues at the active site of Rv2466c form a disulfide bond that is broken down in a mycothiol-dependent manner. This generates a mycothione molecule that is recycled back to two mycothiol molecules through the catalytic activity of Mrx1, an enzyme that utilizes the reducing power of NADPH. The reduced Rv2466c enzyme can then activate nitro prodrugs into active metabolites that kill the bacteria. This also regenerates the oxidized form of the enzyme that can participate in multiple cycles of the reaction.