Structural insights of catalytic mechanism in mutant pyrazinamidase of Mycobacterium tuberculosis.

Pyrazinamidase (PZase) is a member of Fe-dependent amidohydrolases that activates pyrazinamide (PZA) into active pyrazinoic acid (POA). PZA, a nicotinamide analogue, is an essential first-line drug used in Mycobacterium tuberculosis (Mtb) treatment. The active form of PZA, POA, is toxic and potently inhibits the growth of latent Mtb, which makes it possible to shorten the conventional 9-month tuberculosis treatment to 6 months. In this study, an extensive molecular dynamics simulation was carried out to the study the resistance mechanism offered by the three mutations Q10P and D12A and G97D. Our results showed that two regions Gln10-His43, Phe50-Gly75 are profoundly affected by these mutations. Among the three mutations, Q10P and D12A mutations strongly disturb the communication among the catalytic triad (Asp8, Lys98 and Cys138). The oxyanion hole is formed between the backbone nitrogen atoms of A134 and C138 which stabilizes the hydroxyl anion of nicotinamide. The D12A mutation greatly disturbs the oxyanion hole formation followed by the Q10P and G97D. Our results also showed that these mutations destabilize the interaction between Fe2+ ion and Asp49, His51, His57 and His71. The binding pocket analysis showed that these mutations increase the cavity volume, which results in loose binding of PZA. MMGBSA analyzes have shown that these mutations reduce the binding affinity to the PZA drug. Our results may provide useful information for the design of new and effective PZase inhibitors based on structural information of WT and mutant PZases.Communicated by Ramaswamy H. Sarma.

Determining the unbinding events and conserved motions associated with the pyrazinamide release due to resistance mutations of Mycobacterium tuberculosis pyrazinamidase.

Pyrazinamide (PZA) is the only first-line antitubercular drug active against latent Mycobacterium tuberculosis (Mtb). It is activated to pyrazinoic acid by the pncA-encoded pyrazinamidase enzyme (PZase). Despite the emergence of PZA drug resistance, the underlying mechanisms of resistance remain unclear. This study investigated part of these mechanisms by modelling a PZA-bound wild type and 82 mutant PZase structures before applying molecular dynamics (MD) with an accurate Fe2+ cofactor coordination geometry. After observing nanosecond-scale PZA unbinding from several PZase mutants, an algorithm was developed to systematically detect ligand release via centre of mass distances (COM) and ligand average speed calculations, before applying the statistically guided network analysis (SGNA) method to investigate conserved protein motions associated with ligand unbinding. Ligand and cofactor perspectives were also investigated. A conserved pair of lid-destabilising motions was found. These consisted of (1) antiparallel lid and side flap motions; (2) the contractions of a flanking region within the same flap and residue 74 towards the core. Mutations affecting the hinge residues (H51 and H71), nearby residues or L19 were found to destabilise the lid. Additionally, other metal binding site (MBS) mutations delocalised the Fe2+ cofactor, also facilitating lid opening. In the early stages of unbinding, a wider variety of PZA poses were observed, suggesting multiple exit pathways. These findings provide insights into the late events preceding PZA unbinding, which we found to occur in some resistant PZase mutants. Further, the algorithm developed here to identify unbinding events coupled with SGNA can be applicable to other similar problems.

Gibbs Free Energy Calculation of Mutation in PncA and RpsA Associated With Pyrazinamide Resistance.

A central approach for better understanding the forces involved in maintaining protein structures is to investigate the protein folding and thermodynamic properties. The effect of the folding process is often disturbed in mutated states. To explore the dynamic properties behind mutations, molecular dynamic (MD) simulations have been widely performed, especially in unveiling the mechanism of drug failure behind mutation. When comparing wild type (WT) and mutants (MTs), the structural changes along with solvation free energy (SFE), and Gibbs free energy (GFE) are calculated after the MD simulation, to measure the effect of mutations on protein structure. Pyrazinamide (PZA) is one of the first-line drugs, effective against latent Mycobacterium tuberculosis isolates, affecting the global TB control program 2030. Resistance to this drug emerges due to mutations in pncA and rpsA genes, encoding pyrazinamidase (PZase) and ribosomal protein S1 (RpsA) respectively. The question of how the GFE may be a measure of PZase and RpsA stabilities, has been addressed in the current review. The GFE and SFE of MTs have been compared with WT, which were already found to be PZA-resistant. WT structures attained a more stable state in comparison with MTs. The physiological effect of a mutation in PZase and RpsA may be due to the difference in energies. This difference between WT and MTs, depicted through GFE plots, might be useful in predicting the stability and PZA-resistance behind mutation. This study provides useful information for better management of drug resistance, to control the global TB problem.

Characterization of pncA Mutations and Prediction of PZA Resistance in Mycobacterium tuberculosis Clinical Isolates From Chongqing, China.

Pyrazinamide (PZA) is widely used to treat drug-sensitive or multidrug resistance tuberculosis. However, conventional PZA susceptibility tests of clinical isolates are rather difficult because of the requirement of acid pH. Since resistance to pyrazinamide is primary mediated by mutation of pncA, an alternative way of PZA susceptibility test is to analyze the pyrazinamidase activities of Mycobacterium tuberculosis clinical isolates. Therefore, a database containing the full spectrum of pncA mutations along with pyrazinamidase activities will be beneficial. To characterize mutations of pncA in M. tuberculosis from Chongqing, China, the pncA gene was sequenced and analyzed in 465 clinical isolates. A total of 124 types of mutations were identified in 424 drug-resistant isolates, while no mutation was identified in the 31 pan-susceptible isolates. Ninety-four of the 124 mutations had previously been reported, and 30 new mutations were identified. Based on reported literatures, 294 isolates could be predicted resistant to pyrazinamide. Furthermore, pyrazinamidase activities of the 30 new mutations were tested using the Escherichia coli pncA gene knockout strain. The results showed that 24 of these new mutations (28 isolates) led to loss of pyrazinamidase activity and six (8 isolates) of them did not. Taken together, 322 isolates with pncA mutations could be predicted to be PZA resistant among the 424 drug-resistant isolates tested. Analysis of pncA mutations and their effects on pyrazinamidase activity will not only enrich our knowledge of comprehensive pncA mutations related with PZA resistance but also facilitate rapid molecular diagnosis of pyrazinamide resistance in M. tuberculosis.

Structural dynamics behind variants in pyrazinamidase and pyrazinamide resistance.

Pyrazinamide (PZA) is an important component of first-line anti-tuberculosis (anti-TB) drugs. The anti-TB agent is activated into an active form, pyrazinoic acid (POA), by Mycobacterium tuberculosis (MTB) pncA gene encoding pyrazinamidase (PZase). The major cause of PZA-resistance has been associated with mutations in the pncA gene. We have detected several novel mutations including V131F, Q141P, R154T, A170P, and V180F (GeneBank Accession No. MH461111) in the pncA gene of PZA-resistant isolates during PZA drug susceptibility testing followed by pncA gene sequencing. Here, we investigated molecular mechanism of PZA-resistance by comparing the results of experimental and molecular dynamics. The mutants (MTs) and wild type (WT) PZase structures in apo and complex with PZA were subjected to molecular dynamic simulations (MD) at the 40 ns. Multiple factors, including root mean square deviations (RMSD), binding pocket, total energy, dynamic cross correlation, and root mean square fluctuations (RMSF) of MTs and WT were compared. The MTs attained a high deviation and fluctuation compared to WT. Binding pocket volumes of the MTs, were found, lower than the WT, and the docking scores were high than WT while shape complementarity scores were lower than that of the WT. Residual motion in MTs are seemed to be dominant in anti-correlated motion. Mutations at locations, V131F, Q141P, R154T, A170P, and V180F, might be involved in the structural changes, possibly affecting the catalytic property of PZase to convert PZA into POA. Our study provides useful information that will enhance the understanding for better management of TB. AbbreviationsDSTdrug susceptibility testingΔelecelectrostatic energyLJLowenstein-Jensen mediumMGITmycobacterium growth indicator tubesMTsmutantsMDmolecular dynamic simulationsMTBMycobacterium tuberculosisNALC-NaOHN-acetyl-l-cysteine-sodium hydroxideNIHNational Institutes of HealthNPTamount of substance (N), pressure (P) temperature (T)NVTmoles (N), volume (V) temperature (T)PZasepyrazinamidaseΔpspolar solvation energyPTRLProvincial Tuberculosis Reference LaboratoryRMSDroot mean square deviationsRMSFroot mean square fluctuationsΔSASAsolvent accessible surface area energyTBtuberculosisGTotaltotal binding free energyΔvdWVan der Waals energyWTwild typeCommunicated by Ramaswamy H. Sarma.

Pyrazinamide resistance and mutations L19R, R140H, and E144K in Pyrazinamidase of Mycobacterium tuberculosis.

Pyrazinamide (PZA) is an important component of first-line antituberculosis drugs activated by Mycobacterium tuberculosis pyrazinamidase (PZase) into its active form pyrazinoic acid. Mutations in the pncA gene have been recognized as the major cause of PZA resistance. We detected some novel mutations, Leucine19Arginine (L19R), Arginine140Histidine (R140H), and Glutamic acid144 Lysine (E144K), in the pncA gene of PZA-resistant isolates in our wet lab PZA drug susceptibility testing and sequencing. As the molecular mechanism of resistance of these variants has not been reported earlier, we have performed multiple analyses to unveil different mechanisms of resistance because of PZase mutations L19R, R140H, and E144K. The mutants and native PZase structures were subjected to comprehensive computational molecular dynamics (MD) simulations at 100 nanoseconds in apo and drug-bound form. Mutants and native PZase binding pocket were compared to observe the consequence of mutations on the binding pocket size. Hydrogen bonding, Gibbs free energy, and natural ligand Fe +2 effect were also analyzed between native and mutants. A significant variation between native and mutant PZase structure activity was observed. The native PZase protein docking score was found to be the maximum, showing strong binding affinity in comparison with mutants. MD simulations explored the effect of the variants on the biological function of PZase. Hydrogen bonding, metal ion Fe +2 deviation, and fluctuation also seemed to be affected because of the mutations L19R, R140H, and E144K. The variants L19R, R140H, and E144K play a significant role in PZA resistance, altering the overall activity of native PZase, including metal ion Fe +2 displacement and free energy. This study offers valuable evidence for better management of drug-resistant tuberculosis.

Pyrazinamide: the importance of uncovering the mechanisms of action in mycobacteria.

Pyrazinamide (PZA) is still one of the key drugs used in current therapeutic regimens for tuberculosis (TB). Despite its importance for TB therapy, the mode of action of PZA remains unknown. PZA has to be converted to its active form pyrazinoic acid (POA) by the nicotinamidase PncA and is then excreted by an unknown efflux pump. At acidic conditions, POA is protonated to HPOA and is reabsorbed into the cell where it causes cellular damage. For a long time, it has been thought that PZA/POA has no defined target of action, but recent studies have shown that both PZA and POA have several different targets interfering with diverse biochemical pathways, especially in the NAD(+) and energy metabolism. PZA resistance seems to depend not only on a defective pyrazinamidase but is also rather a result of the interplay of many different enzyme targets and transport mechanisms.

pncA gene expression and prediction factors on pyrazinamide resistance in Mycobacterium tuberculosis.

Mutations in the pyrazinamidase (PZAse) coding gene, pncA, have been considered as the main cause of pyrazinamide (PZA) resistance in Mycobacterium tuberculosis. However, recent studies suggest there is no single mechanism of resistance to PZA. The pyrazinoic acid (POA) efflux rate is the basis of the PZA susceptibility Wayne test, and its quantitative measurement has been found to be a highly sensitive and specific predictor of PZA resistance. Based on biological considerations, the POA efflux rate is directly determined by the PZAse activity, the level of pncA expression, and the efficiency of the POA efflux pump system. This study analyzes the individual and the adjusted contribution of PZAse activity, pncA expression and POA efflux rate on PZA resistance. Thirty M. tuberculosis strains with known microbiological PZA susceptibility or resistance were analyzed. For each strain, PZAse was recombinantly produced and its enzymatic activity measured. The level of pncA mRNA was estimated by quantitative RT-PCR, and the POA efflux rate was determined. Mutations in the pncA promoter were detected by DNA sequencing. All factors were evaluated by multiple regression analysis to determine their adjusted effects on the level of PZA resistance. Low level of pncA expression associated to mutations in the pncA promoter region was observed in pncA wild type resistant strains. POA efflux rate was the best predictor after adjusting for the other factors, followed by PZAse activity. These results suggest that tests which rely on pncA mutations or PZAse activity are likely to be less predictive of real PZA resistance than tests which measure the rate of POA efflux. This should be further analyzed in light of the development of alternate assays to determine PZA resistance.

Insight to pyrazinamide resistance in Mycobacterium tuberculosis by molecular docking.

Pyrazinamide (PZA) - an important drug in the anti-tuberculosis therapy, activated by an enzyme Pyrazinamidase (PZase). The basis of PZA resistance in Mycobacterium tuberculosis was owing to mutation in pncA gene coding for PZase. Homology modeling of PZase was performed using software Discovery Studio (DS) 2.0 based on the crystal structure of the PZase from Pyrococcus horikoshii (PDB code 1im5), in this study. The model comprises of one sheet with six parallel strands and seven helices with the amino acids Asp8, Asp49, Trp68, Lys96, Ala134, Thr135 and Cys138 at the active site. Five mutants were generated with Gly at position 8, Thr at position 96, Arg at position 104, Tyr and Ser at position 138. The Wild-type (WT) and five mutant models were docked with PZA. The results indicate that the mutants Lys96Thr, Ser104Arg Asp8Gly and Cys138Tyr may contribute to higher level drug resistance than Cys138Ser. These models provide the first in-silico evidence for the binding interaction of PZA with PZase and form the basis for rationalization of PZA resistance in naturally occurring pncA mutant strains of M. tuberculosis.