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

Abstract

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., FH), 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.

Mycobacterial F420H2-Dependent Reductases Promiscuously Reduce Diverse Compounds through a Common Mechanism

“…Menadione, 3-cyanocoumarin, and 2-cyclohexen-1-one are predicted to be oriented such that their activated alkene groups are within 5 Å of the nucleophilic C5 center of F 420 H - ( Figures 2A–C ). This suggests that, in line with the activity assays ( Figure 1 ) and previously proposed mechanisms (Taylor et al, 2010; Lapalikar et al, 2012b; Ahmed et al, 2015; Mohamed et al, 2016b), catalysis will occur through nucleophilic attack of the C5 hydride to the electrophilic alkene. In the case of malachite green, the alkene moiety (C1 position) of the substrate is 4.2 Å away from C5 of the cofactor, whereas the N,N -dimethylamine and N,N -dimethyliminium moieties point toward the solvent phase ( Figure 2D ).…”

“…All four substrates are predicted to make extensive hydrophobic interactions with aromatic residues in the active site, including a triad of tyrosine residues (Tyr120, Tyr123, Tyr126) that have previously been shown to facilitate hydrophobic shielding during nitroimidazole activation (Mohamed et al, 2016b). There was also evidence of hydrophobic interactions between substrate and cofactor, including different degrees of π-stacking interactions with the isoalloxazine ring ( Figure 2 ).…”

“…These reductases are abundant in mycobacteria and other Actinobacteria, where they have diverged into at least 14 distinct subgroups (A1–A3, B1–B6, AA1–AA5; Ahmed et al, 2015). While several native functions have been proposed, e.g., menaquinone and biliverdin reduction (Gurumurthy et al, 2013; Ahmed et al, 2015, 2016), the enzymes also mediate promiscuous activities, such as nitroimidazole prodrug activation (Cellitti et al, 2012; Mohamed et al, 2016a,b), biodegradation of furanocoumarins (Taylor et al, 2010; Lapalikar et al, 2012b; Jirapanjawat et al, 2016), and decolorization of triarylmethane dyes (Guerra-Lopez et al, 2007; Jirapanjawat et al, 2016). The findings that these enzymes can reduce such structurally and chemically diverse compounds suggests that they may also have the latent capacity to act upon industrially relevant non-natural chemicals.…”

“…The findings that these enzymes can reduce such structurally and chemically diverse compounds suggests that they may also have the latent capacity to act upon industrially relevant non-natural chemicals. Mechanistic studies focused on mycobacteria indicate that these enzymes adopt a distinct mechanism from old yellow enzymes that may be relevant for selective synthesis (Greening et al, 2016; Mohamed et al, 2016b). For example, the reduced cofactor is thought to bind the enzyme from the solvent phase and directly mediate hydride addition to the substrate (Mohamed et al, 2016a,b); The cofactor can then be re-reduced in vitro and in vivo by Fgd (Purwantini and Daniels, 1996; Bashiri et al, 2008).…”

“…Mechanistic studies focused on mycobacteria indicate that these enzymes adopt a distinct mechanism from old yellow enzymes that may be relevant for selective synthesis (Greening et al, 2016; Mohamed et al, 2016b). For example, the reduced cofactor is thought to bind the enzyme from the solvent phase and directly mediate hydride addition to the substrate (Mohamed et al, 2016a,b); The cofactor can then be re-reduced in vitro and in vivo by Fgd (Purwantini and Daniels, 1996; Bashiri et al, 2008). In addition, the proton donor for reduced substrates is a solvent-accessible hydroxonium ion rather than a tyrosine residue (Mohamed et al, 2016b).…”