Pyrazinoic Acid Inhibits Mycobacterial Coenzyme A Biosynthesis by Binding to Aspartate Decarboxylase PanD

Abstract

Previously, we showed that a major in vitro and in vivo mechanism of resistance to pyrazinoic acid (POA), the bioactive component of the critical tuberculosis (TB) prodrug pyrazinamide (PZA), involves missense mutations in the aspartate decarboxylase PanD, an enzyme required for coenzyme A biosynthesis. What is the mechanism of action of POA? Upon demonstrating that treatment of M. bovis BCG with POA resulted in a depletion of intracellular coenzyme A and confirming that this POA-mediated depletion is prevented by either missense mutations in PanD or exogenous supplementation of pantothenate, we hypothesized that POA binds to PanD and that this binding blocks the biosynthetic pathway. Here, we confirm both hypotheses. First, metabolomic analyses showed that POA treatment resulted in a reduction of the concentrations of all coenzyme A precursors downstream of the PanD-mediated catalytic step. Second, using isothermal titration calorimetry, we established that POA, but not its prodrug PZA, binds to PanD. Binding was abolished for mutant PanD proteins. Taken together, these findings support a mechanism of action of POA in which the bioactive component of PZA inhibits coenzyme A biosynthesis via binding to aspartate decarboxylase PanD. Together with previous works, these results establish PanD as a genetically, metabolically, and biophysically validated target of PZA.

Drug resistance mechanisms and drug susceptibility testing for tuberculosis

“…Although there was initial confusion and doubt about PanD as a target of PZA, a more recent study by Gopal et al . confirms the earlier finding that PanD is indeed a target of PZA . In addition, recent studies also identified another possible target of PZA as ClpC1, part of a protease complex involved in protein degradation, which is presumably important for persister survival.…”

“…58,59 Although there was initial confusion and doubt about PanD as a target of PZA, 60 a more recent study by Gopal et al confirms the earlier finding that PanD is indeed a target of PZA. 59,61 In addition, recent studies also identified another possible target of PZA as ClpC1, 62,63 part of a protease complex involved in protein degradation, which is presumably important for persister survival. Furthermore, a recent study identified novel mutations in LprG (rv1411c), rv0521, rv3630, rv0010c, ppsC and cyp128 associated with POA/PZA resistance in MTB, 64 which sheds new light on mode of action and resistance of this intriguing persister drug.…”

Impact of the host environment on the antitubercular action of pyrazinamide

“…In support of PanD as a target of POA, Gopal et al showed binding of POA to PanD (K D = 6.1 μM ± 0.88 μM), which was abrogated with resistant variants [40] . Treatment of wild-type M. bovis BCG with POA was also found to decrease CoA abundance after 24-48 h of exposure [42] , and a mutant strain disrupted in the acyl-CoA ligase FadD2 showed heightened susceptibility to POA [49] .…”

“…Recently, numerous studies have shown that a component of the coenzyme A (CoA) biosynthetic pathway may be a molecular target of POA ( Fig. 1 ) [40][41][42][43][44][45] . Analysis of spontaneous PZA and POA resistant isolates with functional PncA revealed missense mutations in panD that encodes l -aspartate-α-decarboxylase, a rate-limiting step in the CoA biosynthetic pathway [42,44] .…”

Substrate‐induced structural alterations of Mycobacterial mycothione reductase and critical residues involved

“…Such mechanism of oligomer variations is common e.g. for the family of peroxiredoxins, which alter between catalytically active dimers and decamers or even dodecamers depending on redox regulation , the Dengue nonstructural protein NS5 proposed to switch from a monomer to dimer during the process of the replication complex , or the mycobacterial aspartate decarboxylase PanD, required for pantothenate and CoenzymeA (CoA) biosynthesis, described to occur in a tetrameric and octameric state . Furthermore, NADPH induces tetramer formation with inactivation of the human dihydrolipoamide dehydrogenase or causing an increase in activity of the related glutathione reductase of Cyanobacterium and Spirulina maxima have been reported.…”