Evaluation of NAD+-Dependent DNA Ligase of Mycobacteria as a Potential Target for Antibiotics

ABSTRACT Mycobacteria contain genes for several DNA ligases, including ligA, which encodes a NAD+-dependent enzyme that has been postulated to be a target for novel antibacterial compounds. Using a homologous recombination system, direct evidence is presented that wild-type ligA cannot be deleted from the chromosome of Mycobacterium smegmatis. Deletions of native ligA in M. smegmatis could be obtained only after the integration of an extra copy of M. smegmatis or Mycobacterium tuberculosis ligA into the attB site of the chromosome, with expression controlled by chemically inducible promoters. The four ATP-dependent DNA ligases encoded by the M. smegmatis chromosome were unable to replace the function of LigA. Interestingly, the LigA protein from M. smegmatis could be substituted with the NAD+-dependent DNA ligase of Escherichia coli or the ATP-dependent ligase of bacteriophage T4. The conditional mutant strains allowed the analysis of the effect of LigA depletion on the growth of M. smegmatis. The protein level of the conditional mutants was estimated by Western blot analysis using antibodies raised against LigA of M. tuberculosis. This revealed that a strong overproduction or depletion of LigA did not affect the growth or survival of mycobacteria under standard laboratory conditions. In conclusion, although NAD+-dependent DNA ligase is essential for mycobacterial viability, only low levels of protein are required for growth. These findings suggest that very efficient inhibition of enzyme activity would be required if NAD+-dependent DNA ligase is to be useful as an antibiotic target in mycobacteria. The strains developed here will provide useful tools for the evaluation of the efficacy of any appropriate compounds in mycobacteria.

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Evaluation of DNA Primase DnaG as a Potential Target for Antibiotics

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01721-13 ◽

2013 ◽

Vol 58 (3) ◽

pp. 1699-1706 ◽

Cited By ~ 14

Author(s):

Aneta Kuron ◽

Malgorzata Korycka-Machala ◽

Anna Brzostek ◽

Marcin Nowosielski ◽

Aidan Doherty ◽

...

Keyword(s):

Standard Laboratory ◽

Content Type ◽

Growth And Survival ◽

Inducible Promoters ◽

Growth Defects ◽

Laboratory Conditions ◽

Recombination System ◽

Conditional Mutant ◽

Single Deletion ◽

Low Levels

ABSTRACTMycobacteria contain genes for several DNA-dependent RNA primases, includingdnaG, which encodes an essential replication enzyme that has been proposed as a target for antituberculosis compounds. Anin silicoanalysis revealed that mycobacteria also possess archaeo-eukaryotic superfamily primases (AEPs) of unknown function. Using a homologous recombination system, we obtained direct evidence that wild-typednaGcannot be deleted from the chromosome ofMycobacterium smegmatiswithout disrupting viability, even in backgrounds in which mycobacterial AEPs are overexpressed. In contrast, single-deletion AEP mutants or mutants defective for all four identifiedM. smegmatisAEP genes did not exhibit growth defects under standard laboratory conditions. Deletion of nativednaGinM. smegmatiswas tolerated only after the integration of an extra intact copy of theM. smegmatisorMycobacterium tuberculosisdnaGgene, under the control of chemically inducible promoters, into theattBsite of the chromosome.M. tuberculosisandM. smegmatisDnaG proteins were overproduced and purified, and their primase activities were confirmed using radioactive RNA synthesis assays. The enzymes appeared to be sensitive to known inhibitors (suramin and doxorubicin) of DnaG. Notably,M. smegmatisbacilli appeared to be sensitive to doxorubicin and resistant to suramin. The growth and survival of conditional mutant mycobacterial strains in which DnaG was significantly depleted were only slightly affected under standard laboratory conditions. Thus, although DnaG is essential for mycobacterial viability, only low levels of protein are required for growth. This suggests that very efficient inhibition of enzyme activity would be required for mycobacterial DnaG to be useful as an antibiotic target.

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Novel Bacterial NAD+-Dependent DNA Ligase Inhibitors with Broad-Spectrum Activity and Antibacterial EfficacyIn Vivo

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01181-10 ◽

2010 ◽

Vol 55 (3) ◽

pp. 1088-1096 ◽

Cited By ~ 62

Author(s):

Scott D. Mills ◽

Ann E. Eakin ◽

Ed T. Buurman ◽

Joseph V. Newman ◽

Ning Gao ◽

...

Keyword(s):

Broad Spectrum ◽

High Throughput Screening ◽

Bacteriophage T4 ◽

Critical Role ◽

Dna Ligase ◽

Infection Model ◽

Kinetic Measurements ◽

Dna Ligases ◽

Adenosine Analogs

ABSTRACTDNA ligases are indispensable enzymes playing a critical role in DNA replication, recombination, and repair in all living organisms. Bacterial NAD+-dependent DNA ligase (LigA) was evaluated for its potential as a broad-spectrum antibacterial target. A novel class of substituted adenosine analogs was discovered by target-based high-throughput screening (HTS), and these compounds were optimized to render them more effective and selective inhibitors of LigA. The adenosine analogs inhibited the LigA activities ofEscherichia coli,Haemophilus influenzae,Mycoplasma pneumoniae,Streptococcus pneumoniae, andStaphylococcus aureus, with inhibitory activities in the nanomolar range. They were selective for bacterial NAD+-dependent DNA ligases, showing no inhibitory activity against ATP-dependent human DNA ligase 1 or bacteriophage T4 ligase. Enzyme kinetic measurements demonstrated that the compounds bind competitively with NAD+. X-ray crystallography demonstrated that the adenosine analogs bind in the AMP-binding pocket of the LigA adenylation domain. Antibacterial activity was observed against pathogenic Gram-positive and atypical bacteria, such asS. aureus,S. pneumoniae,Streptococcus pyogenes, andM. pneumoniae, as well as against Gram-negative pathogens, such asH. influenzaeandMoraxella catarrhalis. The mode of action was verified using recombinant strains with altered LigA expression, an Okazaki fragment accumulation assay, and the isolation of resistant strains withligAmutations.In vivoefficacy was demonstrated in a murineS. aureusthigh infection model and a murineS. pneumoniaelung infection model. Treatment with the adenosine analogs reduced the bacterial burden (expressed in CFU) in the corresponding infected organ tissue as much as 1,000-fold, thus validating LigA as a target for antibacterial therapy.

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Use of ATP, dATP and their α-thio derivatives to study DNA ligase adenylation

Biochemical Journal ◽

10.1042/bj2710265 ◽

1990 ◽

Vol 271 (1) ◽

pp. 265-268 ◽

Cited By ~ 8

Author(s):

A Montecucco ◽

M Lestingi ◽

G Pedrali-Noy ◽

S Spadari ◽

G Ciarrocchi

Keyword(s):

Bacteriophage T4 ◽

Solid Matrix ◽

Dna Ligase ◽

Type I ◽

Dna Ligases ◽

Human Type ◽

Ligation Reaction ◽

Specific Inhibitors ◽

Covalently Bound

Bacteriophage-T4 and human type I DNA ligases were found capable of self-adenylating upon exposure to both ribo- and deoxyribo-[alpha-35S]thio-ATP. However, the joining reaction does not take place in the presence of the deoxyribotriphosphates. Enzyme adenylation is reversed in all cases by an excess of PPi, but the rate of reversion is lower with thio derivatives. Therefore thio derivatives can be used to study the adenylation of DNA ligases and to search for specific inhibitors of the first step of the ligation reaction. In addition we show that thio derivatives can be used to detect DNA ligase adenylation activity covalently bound to a solid matrix.

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Interaction of bacteriophage T4 RNA and DNA ligases in joining of duplex DNA at base-paired ends.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)40347-4 ◽

1977 ◽

Vol 252 (11) ◽

pp. 3987-3994 ◽

Cited By ~ 4

Author(s):

A Sugino ◽

H M Goodman ◽

H L Heyneker ◽

J Shine ◽

H W Boyer ◽

...

Keyword(s):

Bacteriophage T4 ◽

Duplex Dna ◽

Dna Ligases ◽

Rna And Dna

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A DNA Ligase from a Hyperthermophilic Archaeon with Unique Cofactor Specificity

Journal of Bacteriology ◽

10.1128/jb.182.22.6424-6433.2000 ◽

2000 ◽

Vol 182 (22) ◽

pp. 6424-6433 ◽

Cited By ~ 45

Author(s):

Masaru Nakatani ◽

Satoshi Ezaki ◽

Haruyuki Atomi ◽

Tadayuki Imanaka

Keyword(s):

Biochemical Study ◽

Protein Product ◽

Optimum Concentration ◽

Dna Ligase ◽

Gene Encoding ◽

Hyperthermophilic Archaeon ◽

Dna Ligases ◽

Cofactor Specificity ◽

Thermophilic Archaeon ◽

Optimum Ph

ABSTRACT A gene encoding DNA ligase (ligTk ) from a hyperthermophilic archaeon, Thermococcus kodakaraensisKOD1, has been cloned and sequenced, and its protein product has been characterized. ligTk consists of 1,686 bp, corresponding to a polypeptide of 562 amino acids with a predicted molecular mass of 64,079 Da. Sequence comparison with previously reported DNA ligases and the presence of conserved motifs suggested that Lig Tk was an ATP-dependent DNA ligase. Phylogenetic analysis indicated that Lig Tk was closely related to the ATP-dependent DNA ligase fromMethanobacterium thermoautotrophicum ΔH, a moderate thermophilic archaeon, along with putative DNA ligases fromEuryarchaeota and Crenarchaeota. We expressedligTk in Escherichia coli and purified the recombinant protein. Recombinant Lig Tk was monomeric, as is the case for other DNA ligases. The protein displayed DNA ligase activity in the presence of ATP and Mg2+. The optimum pH of Lig Tk was 8.0, the optimum concentration of Mg2+, which was indispensable for the enzyme activity, was 14 to 18 mM, and the optimum concentration of K+ was 10 to 30 mM. Lig Tk did not display single-stranded DNA ligase activity. At enzyme concentrations of 200 nM, we observed significant DNA ligase activity even at 100°C. Unexpectedly, Lig Tk displayed a relatively small, but significant, DNA ligase activity when NAD+ was added as the cofactor. Treatment of NAD+ with hexokinase did not affect this activity, excluding the possibility of contaminant ATP in the NAD+ solution. This unique cofactor specificity was also supported by the observation of adenylation of Lig Tk with NAD+. This is the first biochemical study of a DNA ligase from a hyperthermophilic archaeon.

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Expression of Mycobacterium smegmatisPyrazinamidase in Mycobacterium tuberculosis Confers Hypersensitivity to Pyrazinamide and Related Amides

Journal of Bacteriology ◽

10.1128/jb.182.19.5479-5485.2000 ◽

2000 ◽

Vol 182 (19) ◽

pp. 5479-5485 ◽

Cited By ~ 34

Author(s):

Helena I. M. Boshoff ◽

Valerie Mizrahi

Keyword(s):

Mycobacterium Tuberculosis ◽

Mycobacterium Smegmatis ◽

Intracellular Localization ◽

Wild Type ◽

Gene Encoding ◽

Allelic Exchange ◽

Mutant Strains ◽

Established Role ◽

Amidase Gene

ABSTRACT A pyrazinamidase (PZase)-deficient pncA mutant ofMycobacterium tuberculosis, constructed by allelic exchange, was used to investigate the effects of heterologous amidase gene expression on the susceptibility of this organism to pyrazinamide (PZA) and related amides. The mutant was highly resistant to PZA (MIC, >2,000 μg/ml), in accordance with the well-established role ofpncA in the PZA susceptibility of M. tuberculosis (A. Scorpio and Y. Zhang, Nat. Med. 2:662–667, 1996). Integration of the pzaA gene encoding the major PZase/nicotinamidase from Mycobacterium smegmatis (H. I. M. Boshoff and V. Mizrahi, J. Bacteriol. 180:5809–5814, 1998) or the M. tuberculosis pncA gene into the pncAmutant complemented its PZase/nicotinamidase defect. In bothpzaA- and pncA-complemented mutant strains, the PZase activity was detected exclusively in the cytoplasm, suggesting an intracellular localization for PzaA and PncA. ThepzaA-complemented strain was hypersensitive to PZA (MIC, ≤10 μg/ml) and nicotinamide (MIC, ≥20 μg/ml) and was also sensitive to benzamide (MIC, 20 μg/ml), unlike the wild-type andpncA-complemented mutant strains, which were highly resistant to this amide (MIC, >500 μg/ml). This finding was consistent with the observation that benzamide is hydrolyzed by PzaA but not by PncA. Overexpression of PzaA also conferred sensitivity to PZA, nicotinamide, and benzamide on M. smegmatis (MIC, 150 μg/ml in all cases) and rendered Escherichia colihypersensitive for growth at low pH.

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MAMMALIAN DNA LIGASES; ALTERED DNA LIGASE I IN BLOOM'S SYNDROME

Radiation Research: A Twentieth-century Perspective ◽

10.1016/b978-0-12-168561-4.50250-x ◽

1991 ◽

pp. 71

Author(s):

ALAN E. TOMKINSON ◽

DEBORAH E. BARNES ◽

KEN-ICHI KODAMA ◽

EEMA ROBERTS ◽

GRAHAM DALY ◽

...

Keyword(s):

Dna Ligase ◽

Bloom's Syndrome ◽

Dna Ligases ◽

Bloom’S Syndrome ◽

Dna Ligase I

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Structural intermediates of a DNA–ligase complex illuminate the role of the catalytic metal ion and mechanism of phosphodiester bond formation

Nucleic Acids Research ◽

10.1093/nar/gkz596 ◽

2019 ◽

Vol 47 (14) ◽

pp. 7147-7162 ◽

Cited By ~ 4

Author(s):

Adele Williamson ◽

Hanna-Kirsti S Leiros

Keyword(s):

Active Site ◽

Metal Ion ◽

Bond Formation ◽

Strand Break ◽

Nucleophilic Attack ◽

Dna Ligase ◽

Phosphodiester Bond ◽

Dna Ligases ◽

Catalytic Metal ◽

Ternary Structure

Abstract DNA ligases join adjacent 5′ phosphate (5′P) and 3′ hydroxyl (3′OH) termini of double-stranded DNA via a three-step mechanism requiring a nucleotide cofactor and divalent metal ion. Although considerable structural detail is available for the first two steps, less is known about step 3 where the DNA-backbone is joined or about the cation role at this step. We have captured high-resolution structures of an adenosine triphosphate (ATP)-dependent DNA ligase from Prochlorococcus marinus including a Mn-bound pre-ternary ligase–DNA complex poised for phosphodiester bond formation, and a post-ternary intermediate retaining product DNA and partially occupied AMP in the active site. The pre-ternary structure unambiguously identifies the binding site of the catalytic metal ion and confirms both its role in activating the 3′OH terminus for nucleophilic attack on the 5′P group and stabilizing the pentavalent transition state. The post-ternary structure indicates that DNA distortion and most enzyme-AMP contacts remain after phosphodiester bond formation, implying loss of covalent linkage to the DNA drives release of AMP, rather than active site rearrangement. Additionally, comparisons of this cyanobacterial DNA ligase with homologs from bacteria and bacteriophage pose interesting questions about the structural origin of double-strand break joining activity and the evolution of these ATP-dependent DNA ligase enzymes.

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The ligation of pol β mismatch insertion products governs the formation of promutagenic base excision DNA repair intermediates

Nucleic Acids Research ◽

10.1093/nar/gkaa151 ◽

2020 ◽

Vol 48 (7) ◽

pp. 3708-3721 ◽

Cited By ~ 4

Author(s):

Melike Çağlayan

Keyword(s):

Excision Repair ◽

Dna Ligase ◽

Active Site Residues ◽

Dna Ligases ◽

Terminal Domain ◽

Dna Ligase I ◽

Nucleotide Insertion ◽

Base Excision ◽

Ligation Step

Abstract DNA ligase I and DNA ligase III/XRCC1 complex catalyze the ultimate ligation step following DNA polymerase (pol) β nucleotide insertion during base excision repair (BER). Pol β Asn279 and Arg283 are the critical active site residues for the differentiation of an incoming nucleotide and a template base and the N-terminal domain of DNA ligase I mediates its interaction with pol β. Here, we show inefficient ligation of pol β insertion products with mismatched or damaged nucleotides, with the exception of a Watson–Crick-like dGTP insertion opposite T, using BER DNA ligases in vitro. Moreover, pol β N279A and R283A mutants deter the ligation of the promutagenic repair intermediates and the presence of N-terminal domain of DNA ligase I in a coupled reaction governs the channeling of the pol β insertion products. Our results demonstrate that the BER DNA ligases are compromised by subtle changes in all 12 possible noncanonical base pairs at the 3′-end of the nicked repair intermediate. These findings contribute to understanding of how the identity of the mismatch affects the substrate channeling of the repair pathway and the mechanism underlying the coordination between pol β and DNA ligase at the final ligation step to maintain the BER efficiency.

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The Redox Cofactor F420Protects Mycobacteria from Diverse Antimicrobial Compounds and Mediates a Reductive Detoxification System

Applied and Environmental Microbiology ◽

10.1128/aem.02500-16 ◽

2016 ◽

Vol 82 (23) ◽

pp. 6810-6818 ◽

Cited By ~ 21

Author(s):

Thanavit Jirapanjawat ◽

Blair Ney ◽

Matthew C. Taylor ◽

Andrew C. Warden ◽

Shahana Afroze ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Mycobacterium Smegmatis ◽

Antimicrobial Compounds ◽

Loss Of Function ◽

Detoxifying Enzymes ◽

Content Type ◽

Wide Range ◽

Detoxification System ◽

Mutant Strains

ABSTRACTA defining feature of mycobacterial redox metabolism is the use of an unusual deazaflavin cofactor, F420. This cofactor enhances the persistence of environmental and pathogenic mycobacteria, including after antimicrobial treatment, although the molecular basis for this remains to be understood. In this work, we explored our hypothesis that F420enhances persistence by serving as a cofactor in antimicrobial-detoxifying enzymes. To test this, we performed a series of phenotypic, biochemical, and analytical chemistry studies in relation to the model soil bacteriumMycobacterium smegmatis. Mutant strains unable to synthesize or reduce F420were found to be more susceptible to a wide range of antibiotic and xenobiotic compounds. Compounds from three classes of antimicrobial compounds traditionally resisted by mycobacteria inhibited the growth of F420mutant strains at subnanomolar concentrations, namely, furanocoumarins (e.g., methoxsalen), arylmethanes (e.g., malachite green), and quinone analogues (e.g., menadione). We demonstrated that promiscuous F420H2-dependent reductases directly reduce these compounds by a mechanism consistent with hydride transfer. Moreover,M. smegmatisstrains unable to make F420H2lost the capacity to reduce and detoxify representatives of the furanocoumarin and arylmethane compound classes in whole-cell assays. In contrast, mutant strains were only slightly more susceptible to clinical antimycobacterials, and this appeared to be due to indirect effects of F420loss of function (e.g., redox imbalance) rather than loss of a detoxification system. Together, these data show that F420enhances antimicrobial resistance in mycobacteria and suggest that one function of the F420H2-dependent reductases is to broaden the range of natural products that mycobacteria and possibly other environmental actinobacteria can reductively detoxify.IMPORTANCEThis study reveals that a unique microbial cofactor, F420, is critical for antimicrobial resistance in the environmental actinobacteriumMycobacterium smegmatis. We show that a superfamily of redox enzymes, the F420H2-dependent reductases, can reduce diverse antimicrobialsin vitroandin vivo.M. smegmatisstrains unable to make or reduce F420become sensitive to inhibition by these antimicrobial compounds. This suggests that mycobacteria have harnessed the unique properties of F420to reduce structurally diverse antimicrobials as part of the antibiotic arms race. The F420H2-dependent reductases that facilitate this process represent a new class of antimicrobial-detoxifying enzymes with potential applications in bioremediation and biocatalysis.

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