Study of pyrazinamidase structural changes in pyrazinamide resistant and susceptible isolates of Mycobacterium tuberculosis

INTRODUCTION
Pyrazinamide is one of the first line four drugs for treatment of tuberculosis. It was proved that mutations in two nucleotides of 359 and 374 pnc genes are highly associated with resistance to pyrazinamide.


MATERIALS AND METHODS
In this study, mutations in these two codones in 30 clinical isolates of Mycobacterium tuberculosis were detected by means of sequencing. Protein structures encoded by this gene with and without mutation were investigated in resistant and susceptible isolates to pyrazinamide, respectively.


RESULTS
Mutation in the positions 359 and 374 altered some parameters like change in electronic charge, distance change of mutated amino acids to situation of active enzyme and metal connection situation. In these conditions, structure and function of pyrozinamidase enzyme were changed and antibiotic was ineffective and consequently caused resistance to pyrazinamide in M. tuberculosis.


CONCLUSION
This work was revealed protein changes in resistance to pyrazinamide in clinical isolates of M. tuberculosis.


INTRODUCTION
Pyrazinamide (PZA) antibiotic is from the nicotinamide analogues which despite is a strong anti bacterial especially in the fairly acidic environment of body microphages and also in zones with acute inflammation.PZA is an analog of nicotinamide and a prodrug that inhibits the growth of Mycobacterium tuberculosis.PZA diffuses into M. tuberculosis cell, where the enzyme pyrazinamidase (PZase) converts PZA to the active form pyrazinoic acid (1).Pyrazinoic acid was thought to inhibit the enzyme fatty acid synthase (FAS) I, which is disrupts membrane potential and interferes with energy production, necessary for survival of M. tuberculosis it is weakly bactericidally effective on the M. tuberculosis yet, it is an efficient drug in the first two-month treatment of tuberculosis, when there are acute inflammation differences in body, and its usage has decreased the treatment duration and the possibility of relapse (3)(4)(5).
PZA requires conversion to the bactericidal compound pyrazinoic acid by the bacterial PZase activity of nicotinamidase to show activity against M. tuberculosis.Mutations leading to a loss of PZase activity cause PZA resistance in M. tuberculosis.Reduction in PZase acti-vity or deletion because of mutation in pnc gene causes resistance to PZA resistance (5,6).
Accumulation of pyrazinoic acid disrupts membrane potential, necessary for survival of M. tuberculosis at an acidic site of infection.Pyrazinoic acid binds to the ribosomal protein S1 (RpsA) and inhibits trans-translation.This may explain the ability of PZA to kill dormant mycobacterium.
Although mutations in any point of pncA gene causes resistance but frequency of mutations in nucleotides of 359 and 374 has been emphasized in literature (7).
The aim of this work was comparison study of protein structure of PZase enzyme in normal and mutated forms.

Bacterial Isolates
In this study, 30 clinical isolates of M. tuberculosis sensitive and resistant to PZA, were selected by proportion method.
PCR products has been sequenced by an ABI system for detection of any mutations in two nucleotides of 359 and 374.

Bioinformatics Analysis
The three dimensional (3-D) structure of the protein coded by pnc-A gene has been evaluated by an informatics investigation using Molegro Virtual Docker (MVD) software.

Molecular Study
PCR amplification on purified DNA of 30 isolates revealed 774 bp (base pair) including the entire ORF of pncA gene (400 bp) plus a part of downstream and upstream of this gene.This confirmed correct conditions and performance of PCR reaction by pnc-8 and pnc-11 primers for amplification (Figure 1).
Results from sequencing of 744-bp fragment in all isolates revealed that susceptible isolates to PZA have any mutations in nucleotides of 359 and 374.Some of resistant isolates to PZA without any mutations in these nucleotides had mutations in other nucleotides.

Protein Study
Comparing wild-type and minimized mutant structures with two codons of 120 and 125, which have respectively been created by changing T to C in 359 nucleotide and T to G in 374 nucleotide, confirms the differences in two predicted structures (Figure 2-4).
Mutated protein structures of changed amino acids show the importance of mutation in studied nucleotide.In nucleotide 359, after T changing to C, the Hydrophobic amino acid No. 120 (Leu) changes to amino acid with special side chain that named proline, and in nucleotide 374, after T changing to G, amino acids No. 125 [Val] changes to Gly.Software analysis by Molegro virtual docker showed that 3-D structure of pyrazinamidase enzyme (PZAase) consists of four alpha helix and four B-sheets (Figure 5).The special arrangement of these motifs creates a hole in the molecule that, along with Fe 2+ ion, is necessary for the activity of the enzyme.This ion plays a direct, catalytic role in hydrolyze of the medicine.The important amino acid residues in performance of PZAase include residues in the enzyme active situation (D8, A134, and C138) and amino acid residues in the metal connection situation (MCS) (D49, H51, H58, and H71) (1,8).Mutation in these amino acids and other amino acids present near these situations has effect on the physicochemical activity of this enzyme.So far, in different studies, after mutation, different parameters have been attended like change in electronic charge and volume of mutated amino acids, distance change of mutated amino acids compared to situation of active enzyme and metal connection situation, and direction of side-chain of mutated amino acid (8,9).In this paper, situation of codon 120 of three amino acids was found to be at the end of one of the helixes, and after the mutation in this protein, another small hole is created.This last one cause a distance change of proline compared to active enzyme situation and metal connection situation and direction of side-chain of mutated amino acid.But in situation of codon 125, direction of sidechain of valine changes compared to wild-type, after this amino acid changes to glycine.With relation between structure and performance of biological macromolecules, this change causes decrease or elimination of enzyme activity, and it can consequently cause resistance to pyrazinomidase (1,10).For example distance between wild type amino acid at 120 (leucine) to active site (cavity) from 17.098 nm changed to 18.1044 nm in mutant amino acid in this situation and so on.

CONCLUSION
In this study, the structures of wild and mutated pyrazinamidases from clinical isolates M. tuberculosis were analyzed and single amino acid replacement was    studied.Mutations in nucleotides 359 and/or 374 from clinical isolates sensitive and resistant to pyrazinamidase were proved by sequencing.Bioinformatics analysis revealed that in the mutant protein structure of the pyrazinamidase enzyme was changed some parameters such as electrical charge of the mutated amino acid, the volume of the mutated amino acid, the distance of the mutated amino acid to the active site, the distance of the mutated amino acid to the metal-coordination site, and the orientation of the side-chain of the mutated amino acid.In situation of codon 125 or 120, direction of side-chain of mutated amino acid and distances to active site changes compared to wild-type.As for relation between structure and performance of enzyme, these changes causes decrease or elimination of enzyme activity, and it can consequently cause resistance to pyrazinamidase.Therefore the antibiotic will be ineffective on the bacterium.

Figure 2 .
Figure 2. Comparison of the predicted two structures wild-type and mutant (L120P) by using molegro virtual docker software: these images showed that changing in situation of codon 120 created another small cavity and distance of proline to active enzyme situation (18.1044 nm) and metal connection situation (20.7969 nm) changed.These parameters at wild-type structures are 17.098 nm and 19.5774 nm.

Figure 3 .
Figure 3.Comparison of the predicted two structures wild-type and mutant (V125G) by molegro virtual docker software: with occurrance a mutation at amino acid No. 125, distance to enzyme active site from 19.9544 nm in wild-type structure is converted to 23.0768 nm in mutant (mut) structure and distance the situation 125 to the metal ion position from the 24.1225 nm in wild-type is converted to 26.6282 nm in mut structure.

Figure 4 .
Figure 4. Comparison of the predicted two structures wild-type and mutant (L 120P and V125G) by molegro virtual docker software: in mutant structure (L120P and V125G) of this protein is created a extended new cavity.

Figure 5 .
Figure 5. Schematic view of three-dimensional structure of pyrazinamidase: as shown the most of superficial regions of the protein are acidic areas (gray colour in the image) and sections marked with an arrow represents the cavity.