Structural analysis of the housecleaning nucleoside triphosphate pyrophosphohydrolase MazG from Mycobacterium tuberculosis.

The housecleaning enzyme of Mycobacterium tuberculosis (Mtb), MazG, is a nucleoside triphosphate pyrophosphohydrolase (NTP-PPase) and can hydrolyze all canonical or non-canonical NTPs into NMPs and pyrophosphate. The Mycobacterium tuberculosis MazG (Mtb-MazG) contributes to antibiotic resistance in response to oxidative or nitrosative stress under dormancy, making it a promising target for treating TB in latent infection patients. However, the structural basis of Mtb-MazG is not clear. Here we describe the crystal structure of Mtb-MazG (1-185) at 2.7 Å resolution, composed of two similar folded spherical domains in tandem. Unlike other all-α NTP pyrophosphatases, Mtb-MazG has an N-terminal extra region composed of three α-helices and five ß-strands. The second domain is global, with five α-helices located in the N-terminal domain. Gel-filtration assay and SAXS analysis show that Mtb-MazG forms an enzyme-active dimer in solution. In addition, the metal ion Mg2+ is bound with four negative-charged residues Glu119, Glu122, Glu138, and Asp141. Different truncations and site-directed mutagenesis revealed that the full-length dimeric form and the metal ion Mg2+ are indispensable for the catalytic activity of Mtb-MazG. Thus, our work provides new insights into understanding the molecular basis of Mtb-MazG.

Staphylococcus aureus MazG hydrolyzes oxidized guanine nucleotides and contributes to oxidative stress resistance.

We previously reported that knockout of the mazG (SA1292) gene decreases Staphylococcus aureus killing activity against silkworms. S. aureus MazG (SaMazG) has a nucleotide pyrophosphatase domain conserved among MazG family proteins, but its biochemical characteristics are unknown. In the present study, we purified recombinant N-terminal His-tagged SaMazG protein and examined its biochemical activity. SaMazG hydrolyzed GTP, UTP, dGTP, and TTP into nucleoside monophosphates. Hydrolytic activity of SaMazG against ATP, CTP, dATP, and dCTP was low or not detected. SaMazG exhibited high hydrolytic activity against 8-oxo-GTP and 8-oxo-dGTP, oxidized guanine nucleotides, with a Vmax/Km ratio more than 15-fold that of GTP. Furthermore, the S. aureus mazG knockout mutant was sensitive to hydrogen peroxide compared with the parent strain. These results suggest that SaMazG is a nucleotide pyrophosphatase hydrolyzing oxidized guanine nucleotides that contributes to the oxidative stress resistance of S. aureus.

The mazG gene involvement in the stringent response of Escherichia coli regulated by mazEF / 国际药学研究杂志

Objective: To explore the molecular mechanism of mazG gene involved in regulating mazEF toxin-antitoxin system(TAs)mediated bacterial growth inhibition and programmed death, and to clarify the true physiological function of MazG protein. Methods: The Escherichia coli (E.coli)strain MC4100 was used as a prototype and the relA gene was recovered to obtain the relA wildtype strain MC4200. E. coli mazG, mazEF, mazEFG and other series of gene knockout strains were constructed to test the effects of mazG gene overexpression in different genetic background strains on the survival rate of bacteria. Rifampicin, H2O2 and nalidixic acid and other stress conditions were used to treat the bacteria and study growth curve and survival rate of mazG gene and mazEFG operondeleted strains. The E.coli mazG gene and mutant were cloned into an inducible overexpression to construct pET28a-mazG and pET28amazG E38A. Then the protein was overexpressed in BL21 strain and purified using Ni-NTA resin. The dephosphatase activity of MazG protein was verified by enzyme experiments and the effect of mutant overexpression on bacterial survival was tested. Results: The overexpression of mazG had no significant effect on the growth of E.coli MC4200, but had a significant inhibitory effect on the mazEFG gene knockout strain. The cytotoxicity of MazG depended on its NTP-PPase enzyme activity. The presence of mazEF significantly inhibited the phenotype of mazG;Knockout of the mazEF/mazG/mazEFG genes did not affect the growth curve of E.coli under normal envi- ronment. Under stress conditions, the survival rate of the mazG knockout strain was basically the same as that of the mazEFG knockout strain, which was significantly higher than that of the wild type. Conclusion: The mazG gene is involved in the regulation of bacterial programmed cell death induced by mazEF and has an important role in bacterial growth inhibition. This study provides a new perspective for the study of TAs and further understanding of its role in the regulation of bacterial growth and death.

Housecleaning of pyrimidine nucleotide pool coordinates metabolic adaptation of nongrowing Mycobacterium tuberculosis.

The ability of Mycobacterium tuberculosis (Mtb) to adopt a slowly growing or nongrowing state within the host plays a critical role for the bacilli to persist in the face of a prolonged multidrug therapy, establish latency and sustain chronic infection. In our previous study, we revealed that genome maintenance via MazG-mediated elimination of oxidized dCTP contributes to the antibiotic tolerance of nongrowing Mtb. Here, we provide evidence that housecleaning of pyrimidine nucleotide pool via MazG coordinates metabolic adaptation of Mtb to nongrowing state. We found that the ΔmazG mutant fails to maintain a nongrowing and metabolic quiescence state under dormancy models in vitro. To investigate bacterial metabolic changes during infection, we employed RNA-seq to compare the global transcriptional response of wild-type Mtb and the ΔmazG mutant after infection of macrophages. Pathway enrichment analyses of the differentially regulated genes indicate that the deletion of mazG in Mtb not only results in DNA instability, but also perturbs pyrimidine metabolism, iron and carbon source uptake, catabolism of propionate and TCA cycle. Moreover, these transcriptional signatures reflect anticipatory metabolism and regulatory activities observed during cell cycle re-entry in the ΔmazG mutant. Taken together, these results provide evidence that pyrimidine metabolism is a metabolic checkpoint during mycobacterial adaptation to nongrowing state.

Deinococcus radiodurans DR2231 is a two-metal-ion mechanism hydrolase with exclusive activity on dUTP.

DR2231 from Deinococcus radiodurans was previously functionally and structurally characterized as an all-α NTP pyrophosphohydrolase with specific dUTPase activity. dUTPases have a central role in the regulation of dUTP intracellular levels and dTTP nucleotide metabolism. DR2231 presents a conserved dimetal catalytic site, similar to all-α dimeric dUTPases, but contrary to these enzymes, it is unable to process dUDP. In this article, we present functional and structural evidence of single-point mutations that affect directly or indirectly the enzyme catalysis and provide a complete description of the all-α NTP pyrophosphohydrolase mechanism. Activity assays, isothermal titration calorimetry and the crystal structures of these mutants obtained in complex with dUMP or a dUTP analogue aid in probing the reaction mechanism. Our results demonstrate that the two metals are necessary for enzyme processing and also important to modulate the substrate binding affinity. Single-point mutations located in a structurally mobile lid-like loop show that the interactions with the nucleoside monophosphate are essential for induction of the closed conformation and ultimately for substrate processing. ß- and γ-phosphates are held in place through coordination with the second metal, which is responsible for the substrate 'gauche' orientation in the catalytic position. The lack of sufficient contacts to orient the dUDP ß-phosphate for hydrolysis explains DR2231 preference towards dUTP. Sequence and structural similarities with MazG proteins suggest that a similar mechanism might be conserved within the protein family. DATABASE: Structural data are available in the PDB under the accession numbers 5HVA, 5HWU, 5HX1, 5HYL, 5I0J, 5HZZ, 5I0M.

Escherichia coli MazEF toxin-antitoxin system does not mediate programmed cell death.

Toxin-antitoxins systems (TAS) are prokaryotic operons containing two small overlapping genes which encode two components referred to as toxin and antitoxin. Involvement of TAS in bacterial programmed cell death (PCD) is highly controversial. MazEF, a typical type II TAS, is particularly implicated in mediating PCD in Escherichia coli. Hence, we compared the metabolic fitness and stress tolerance of E. coli strains (MC4100 and its mazEF-derivative) which were extensively used by proponents of mazEF-mediated PCD. We found that both the strains are deficient in relA gene and that the ΔmazEF strain has lower fitness and stress tolerance compared to wild type MC4100. We could not reproduce mazEF mediated PCD which emphasizes the need for skeptic approach to the PCD hypothesis.

Crystal structure of the Bacillus-conserved MazG protein, a nucleotide pyrophosphohydrolase.

BA1544 from Bacillus anthracis was previously annotated as a transcription factor for the gene cluster ba1554 - ba1558, but has not been experimentally characterized. B. anthracis is an obligate pathogen causing fatal inhalational anthrax, and BA1544 is absolutely conserved in Bacillus species, including Bacillus cereus, Bacillus thuringiensis and Bacillus mycoides, with 100% sequence identity. To address the function of BA1544, we performed structural and biochemical studies, which revealed that BA1544 is a MazG protein. Thus, herein, the protein is defined as Bacillus-conserved MazG (BcMazG). Like other MazG structures, BcMazG assembles into a tetrameric architecture. Each monomer adopts a four-α-helix bundle that accommodates a metal ion using four acidic residues, and presents one putative substrate-binding site. Enzymatic characterization demonstrated that BcMazG is a nucleoside triphosphate (NTP) pyrophosphohydrolase and prefers adenosine triphosphate as a substrate among canonical NTPs. Moreover, structural comparison of BcMazG with its homologues revealed a potential regulation mechanism whereby the enzymatic activity of BcMazG is regulated by its C-terminal region.