Bacterial Type IIA Topoisomerases Research Articles

How Do Gepotidacin and Zoliflodacin Stabilize DNA-Cleavage Complexes with Bacterial Type IIA Topoisomerases? 2. A Single Moving Metal Mechanism.

DNA gyrase is a bacterial type IIA topoisomerase that can create temporary double-stranded DNA breaks to regulate DNA topology and an archetypical target of antibiotics. The widely used quinolone class of drugs use a water-metal ion bridge in interacting with the GyrA subunit of DNA gyrase. Zoliflodacin sits in the same pocket as quinolones but interacts with the GyrB subunit and also stabilizes lethal double-stranded DNA breaks. Gepotidacin has been observed to sit on the twofold axis of the complex, midway between the two four-base-pair separated DNA-cleavage sites and has been observed to stabilize singe-stranded DNA breaks. Here, we use information from three crystal structures of complexes of Staphlococcus aureus DNA gyrase (one with a precursor of gepotidacin and one with the progenitor of zoliflodacin) to propose a simple single moving metal-ion-catalyzed DNA-cleavage mechanism. Our model explains why the catalytic tyrosine is in the tyrosinate (negatively charged) form for DNA cleavage. Movement of a single catalytic metal-ion (Mg2+ or Mn2+) guides water-mediated protonation and cleavage of the scissile phosphate, which is then accepted by the catalytic tyrosinate. Type IIA topoisomerases need to be able to rapidly cut the DNA when it becomes positively supercoiled (in front of replication forks and transcription bubbles) and we propose that the original purpose of the small Greek Key domain, common to all type IIA topoisomerases, was to allow access of the catalytic metal to the DNA-cleavage site. Although the proposed mechanism is consistent with published data, it is not proven and other mechanisms have been proposed. Finally, how such mechanisms can be experimentally distinguished is considered.

How Do Gepotidacin and Zoliflodacin Stabilize DNA Cleavage Complexes with Bacterial Type IIA Topoisomerases? 1. Experimental Definition of Metal Binding Sites.

One of the challenges for experimental structural biology in the 21st century is to see chemical reactions happen. Staphylococcus aureus (S. aureus) DNA gyrase is a type IIA topoisomerase that can create temporary double-stranded DNA breaks to regulate DNA topology. Drugs, such as gepotidacin, zoliflodacin and the quinolone moxifloxacin, can stabilize these normally transient DNA strand breaks and kill bacteria. Crystal structures of uncleaved DNA with a gepotidacin precursor (2.1 Å GSK2999423) or with doubly cleaved DNA and zoliflodacin (or with its progenitor QPT-1) have been solved in the same P61 space-group (a = b ≈ 93 Å, c ≈ 412 Å). This suggests that it may be possible to observe the two DNA cleavage steps (and two DNA-religation steps) in this P61 space-group. Here, a 2.58 Å anomalous manganese dataset in this crystal form is solved, and four previous crystal structures (1.98 Å, 2.1 Å, 2.5 Å and 2.65 Å) in this crystal form are re-refined to clarify crystal contacts. The structures clearly suggest a single moving metal mechanism-presented in an accompanying (second) paper. A previously published 2.98 Å structure of a yeast topoisomerase II, which has static disorder around a crystallographic twofold axis, was published as containing two metals at one active site. Re-refined coordinates of this 2.98 Å yeast structure are consistent with other type IIA topoisomerase structures in only having one metal ion at each of the two different active sites.

In Silico Activity Prediction and Docking Studies of the Binding Mechanisms of Levofloxacin Structure Derivatives to Active Receptor Sites of Bacterial Type IIA Topoisomerases

The need for new antimicrobial agents (AntAg) is driven by the persistent antibiotic resistance in microorganisms, as well as the increasing frequency of pandemics. Due to the deficiency of AntAg, research aimed at developing speedy approaches to find new drug candidates is relevant. This study aims to conduct an in silico study of the biological activity spectrum as well as the molecular binding mechanisms of four structurally different forms of levofloxacin (Lvf) with bacterial topoisomerases targets of type IIA (DNA gyrase and topoisomerase IV) to enable the development of drugs with an improved characterization of the safety profile. To achieve this goal, a number of software products were used, such as ChemicPen v. 2.6, PyMol 2.5, Avogadro 1.2.0, PASS, AutoDockTools 1.5.7 with the new generation software Autodock Vina. These software products are the first to be made available for visualization of clusters with determination of ligand-receptor pair binding affinity, as well as clustering coordinates and proposed mechanisms of action. One of the real structures of Lvf, a decarboxylated derivative, was obtained with tribochemical (TrbCh) exposure. The action spectrum of molecular ligands is described based on a Bayesian probability activity prediction model (PASS software Version 2.0). Predicted and real (PMS and RMS) molecular structures of Lvf, with decreasing levels of structural complexity, were translated into descriptors via Wiener (W), Balaban (Vs), Detour (Ip), and Electropy € indices. The 2D «structure-activity» diagrams were used to differentiate closely related structures of levofloxacin. PMS and RMS were visualized as 3D models of the ligand-receptor complexes. The contact regions of RMS and PMS with key amino acid residues—SER-79, DT-15, DG-1, DA-1—were demonstrated. The intra- and inter-molecular binding sites, data on free energy (affinity values, kcal/mol), the binding constant Kb (M−1), and the number of clusters are presented. The research results obtained from the presented in silico approach to explore the spectrum of action find quantitative “structure-activity” correlations, and predict molecular mechanisms may be of applied interest for directed drug discovery.

A 2.8 Å Structure of Zoliflodacin in a DNA Cleavage Complex with Staphylococcus aureus DNA Gyrase.

Since 2000, some thirteen quinolones and fluoroquinolones have been developed and have come to market. The quinolones, one of the most successful classes of antibacterial drugs, stabilize DNA cleavage complexes with DNA gyrase and topoisomerase IV (topo IV), the two bacterial type IIA topoisomerases. The dual targeting of gyrase and topo IV helps decrease the likelihood of resistance developing. Here, we report on a 2.8 Å X-ray crystal structure, which shows that zoliflodacin, a spiropyrimidinetrione antibiotic, binds in the same DNA cleavage site(s) as quinolones, sterically blocking DNA religation. The structure shows that zoliflodacin interacts with highly conserved residues on GyrB (and does not use the quinolone water-metal ion bridge to GyrA), suggesting it may be more difficult for bacteria to develop target mediated resistance. We show that zoliflodacin has an MIC of 4 µg/mL against Acinetobacter baumannii (A. baumannii), an improvement of four-fold over its progenitor QPT-1. The current phase III clinical trial of zoliflodacin for gonorrhea is due to be read out in 2023. Zoliflodacin, together with the unrelated novel bacterial topoisomerase inhibitor gepotidacin, is likely to become the first entirely novel chemical entities approved against Gram-negative bacteria in the 21st century. Zoliflodacin may also become the progenitor of a new safer class of antibacterial drugs against other problematic Gram-negative bacteria.

Thiosemicarbazide Derivatives Decrease the ATPase Activity of Staphylococcus aureus Topoisomerase IV, Inhibit Mycobacterial Growth, and Affect Replication in Mycobacterium smegmatis

Compounds targeting bacterial topoisomerases are of interest for the development of antibacterial agents. Our previous studies culminated in the synthesis and characterization of small-molecular weight thiosemicarbazides as the initial prototypes of a novel class of gyrase and topoisomerase IV inhibitors. To expand these findings with further details on the mode of action of the most potent compounds, enzymatic studies combined with a molecular docking approach were carried out, the results of which are presented herein. The biochemical assay for 1-(indol-2-oyl)-4-(4-nitrophenyl) thiosemicarbazide (4) and 4-benzoyl-1-(indol-2-oyl) thiosemicarbazide (7), showing strong inhibitory activity against Staphylococcus aureus topoisomerase IV, confirmed that these compounds reduce the ability of the ParE subunit to hydrolyze ATP rather than act by stabilizing the cleavage complex. Compound 7 showed better antibacterial activity than compound 4 against clinical strains of S. aureus and representatives of the Mycobacterium genus. In vivo studies using time-lapse microfluidic microscopy, which allowed for the monitoring of fluorescently labelled replisomes, revealed that compound 7 caused an extension of the replication process duration in Mycobacterium smegmatis, as well as the growth arrest of bacterial cells. Despite some similarities to the mechanism of action of novobiocin, these compounds show additional, unique properties, and can thus be considered a novel group of inhibitors of the ATPase activity of bacterial type IIA topoisomerases.

Towards Conformation-Sensitive Inhibition of Gyrase: Implications of Mechanistic Insight for the Identification and Improvement of Inhibitors.

Gyrase is a bacterial type IIA topoisomerase that catalyzes negative supercoiling of DNA. The enzyme is essential in bacteria and is a validated drug target in the treatment of bacterial infections. Inhibition of gyrase activity is achieved by competitive inhibitors that interfere with ATP- or DNA-binding, or by gyrase poisons that stabilize cleavage complexes of gyrase covalently bound to the DNA, leading to double-strand breaks and cell death. Many of the current inhibitors suffer from severe side effects, while others rapidly lose their antibiotic activity due to resistance mutations, generating an unmet medical need for novel, improved gyrase inhibitors. DNA supercoiling by gyrase is associated with a series of nucleotide- and DNA-induced conformational changes, yet the full potential of interfering with these conformational changes as a strategy to identify novel, improved gyrase inhibitors has not been explored so far. This review highlights recent insights into the mechanism of DNA supercoiling by gyrase and illustrates the implications for the identification and development of conformation-sensitive and allosteric inhibitors.

Synthetic and Biological Studies on New Urea and Triazole Containing Cystobactamid Derivatives.

Cystobactamids belong to the group of arene‐based oligoamides that effectively inhibit bacterial type IIa topoisomerases. Cystobactamid 861‐2 is the most active member of these antibiotics. Most amide bonds present in the cystobactamids link benzoic acids with anilines and it was found that some of these amide bonds undergo chemical and enzymatic hydrolysis, especially the one linking ring C with ring D. This work reports on the chemical synthesis and biological evaluation of thirteen new cystobactamids that still contain the methoxyaspartate hinge. However, we exchanged selected amide bonds either by the urea or the triazole groups and modified ring A in the latter case. While hydrolytic stability could be improved with these structural substitutes, the high antibacterial potency of cystobactamid 861‐2 could only be preserved in selected cases. This includes derivatives, in which the urea group is positioned between rings A and B and where the triazole is found between rings C and D.

Novel N-1 substituted fluoroquinolones inhibit human topoisomerase I activity and exhibit anti-proliferative activity.

Fluoroquinolone-class agents selectively target the bacterial type IIA topoisomerases DNA gyrase and topoisomerase IV, with a few exceptions that target eukaryotic type IIA topoisomerases. Fluoroquinolones bind and stabilize type IIA topoisomerase-DNA covalent complexes that contain a double-strand break. This unique mode of action is referred to as 'topoisomerase poisoning'. We discovered that two novel fluoroquinolones having aryl functionality at the N-1 position, UITT-3-217 (217) and UITT-3-227 (227), could inhibit the catalytic activity of human topoisomerase II without stabilizing topoisomerase-DNA complexes, i.e., without poisoning it. Surprisingly, these compounds are more effective in inhibiting the catalytic activities of human and bacterial topoisomerase I. The National Cancer Institute's 60 human tumor cell lines screen revealed significant anti-proliferative activities with 217 and 227 against the majority of 60 cancer cell lines. A proof of concept in vivo efficacy study using an HT-29 xenograft model of human colorectal cancer showed that 217 could inhibit the proliferation of human colorectal cancer cells to a degree comparable to fluorouracil in mice. Although 227 also exhibited anti-proliferative activity, it was not as effective as 217 in this xenograft model. These novel fluoroquinolones may serve as promising lead compounds for the development of new anticancer drugs.

Novel tricyclics (e.g., GSK945237) as potent inhibitors of bacterial type IIA topoisomerases

During the course of our research on the lead optimisation of the NBTI (Novel Bacterial Type II Topoisomerase Inhibitors) class of antibacterials, we discovered a series of tricyclic compounds that showed good Gram-positive and Gram-negative potency. Herein we will discuss the various subunits that were investigated in this series and report advanced studies on compound 1 (GSK945237) which demonstrates good PK and in vivo efficacy properties.

Biological profiling of novel tricyclic inhibitors of bacterial DNA gyrase and topoisomerase IV.

The objective of this study was to characterize the in vitro and in vivo biological properties of a novel series of small-molecule bacterial type IIA topoisomerase inhibitors. Bacterial susceptibility testing was performed by broth microdilution. Resistance frequencies were determined by plating bacteria onto agar containing test compound and enumerating mutants. Bacteria were passaged using subinhibitory concentrations of antibacterials to generate resistance. Target enzyme inhibition was determined by exposure to antibacterials and DNA; topoisomers were visualized by gel electrophoresis. Oral and intravenous pharmacokinetic profiles were determined in mice. In vivo efficacy was determined using a mouse model of septicaemia and thigh infection with MSSA and MRSA, respectively. Representative compounds REDX04139, REDX05604 and REDX05931 demonstrated in vitro potency against a range of Gram-positive and fastidious Gram-negative pathogens. Clinical isolate testing revealed REDX04139 and REDX05931 had MIC90 values of 0.25 and 0.5 mg/L, respectively, for MRSA and MIC90 values of 2 mg/L for streptococci. REDX04139 was bactericidal in vitro against Staphylococcus aureus at 8× MIC over 6 h. Pharmacokinetic profiling of REDX04139 and REDX05604 in mice revealed low clearance and excellent bioavailability (≥71%). REDX04139 provided 100% survival against S. aureus in a mouse septicaemia model, while REDX05604 reduced bacterial load by up to 3.7 log units in the MRSA mouse thigh infection model. Redx Pharma has discovered a novel series of topoisomerase inhibitors that are being further developed for drug-resistant bacteria.

Synthesis and evaluation of original bioisosteres of bacterial type IIA topoisomerases inhibitors

A recently discovered series of inhibitors of the ATPase function of bacterial type IIA topoisomerases featuring a carboxypyrrole component led us to attempt to replace this group with a potentially bioisosteric carboxypyrazole. Accordingly, synthetic pathways to 2-(4-(1H-pyrazole-5-carboxamido)piperidin-1-yl)thiazole-5-carboxylic acids or 2-(4-(N-methyl-1H-pyrazole-5-carboxamido)piperidin-1-yl)thiazole-5-carboxylic acids featuring an array of substituents on the pyrazole ring were explored. Unfortunately, none of the analogues made were effective on the ATPase function of Mycobacterium tuberculosis gyrase as well on the DNA supercoiling activity of the whole gyrase of M. tuberculosis and Escherichia coli. However, this work is still providing original insights in chemistry as well as in the structure–activity relationships of this series of inhibitors.

Fluoroquinolones stimulate the DNA cleavage activity of topoisomerase IV by promoting the binding of Mg2 + to the second metal binding site

BackgroundFluoroquinolones target bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV (Topo IV). Fluoroquinolones trap a topoisomerase–DNA covalent complex as a topoisomerase–fluoroquinolone–DNA ternary complex and ternary complex formation is critical for their cytotoxicity. A divalent metal ion is required for type IIA topoisomerase-catalyzed strand breakage and religation reactions. Recent studies have suggested that type IIA topoisomerases use two metal ions, one structural and one catalytic, to carry out the strand breakage reaction. MethodsWe conducted a series of DNA cleavage assays to examine the effects of fluoroquinolones and quinazolinediones on Mg2+-, Mn2+-, or Ca2+-supported DNA cleavage activity of Escherichia coli Topo IV. ResultsIn the absence of any drug, 20–30mMMg2+ was required for the maximum levels of the DNA cleavage activity of Topo IV, whereas approximately 1mM of either Mn2+ or Ca2+ was sufficient to support the maximum levels of the DNA cleavage activity of Topo IV. Fluoroquinolones promoted the Topo IV-catalyzed strand breakage reaction at low Mg2+ concentrations where Topo IV alone could not efficiently cleave DNA. Conclusions and general significanceAt low Mg2+ concentrations, fluoroquinolones may stimulate the Topo IV-catalyzed strand breakage reaction by promoting Mg2+ binding to metal binding site B through the structural distortion in DNA. As Mg2+ concentration increases, fluoroquinolones may inhibit the religation reaction by either stabilizing Mg2+ at site B or inhibition the binding of Mg2+ to site A. This study provides a molecular basis of how fluoroquinolones stimulate the Topo IV-catalyzed strand breakage reaction by modulating Mg2+ binding.

Crystallization and initial crystallographic analysis of covalent DNA-cleavage complexes of Staphyloccocus aureus DNA gyrase with QPT-1, moxifloxacin and etoposide.

Fluoroquinolone drugs such as moxifloxacin kill bacteria by stabilizing the normally transient double-stranded DNA breaks created by bacterial type IIA topoisomerases. Previous crystal structures of Staphylococcus aureus DNA gyrase with asymmetric DNAs have had static disorder (with the DNA duplex observed in two orientations related by the pseudo-twofold axis of the complex). Here, 20-base-pair DNA homoduplexes were used to obtain crystals of covalent DNA-cleavage complexes of S. aureus DNA gyrase. Crystals with QPT-1, moxifloxacin or etoposide diffracted to between 2.45 and 3.15 Å resolution. A G/T mismatch introduced at the ends of the DNA duplexes facilitated the crystallization of slightly asymmetric complexes of the inherently flexible DNA-cleavage complexes.

Biological evaluation and molecular modelling study of thiosemicarbazide derivatives as bacterial type IIA topoisomerases inhibitors

In the present article, we describe the inhibitory potency of nine thiosemicarbazide derivatives against bacterial type IIA topoisomerases, their antibacterial profile and molecular modelling evaluation. We found that one of the tested compounds, compound 7, significantly inhibits activity of Staphylococcus aureus DNA gyrase with an IC50 below 15 μM. Besides, this compound displays antibacterial activity on reference Staphylococuss spp. and Enterococcus faecalis strains as well as clinical S. aureus isolates at non-cytotoxic concentrations in mammalian cells with MIC values ranging from 16 to 32 μg/mL thereby indicating, in some cases, equipotent or even more effective action than standard drugs such as vancomycin, ampicillin and nitrofurantoin. The computational studies showed that both molecular geometry and the electron density distribution have a great impact on antibacterial activity of thiosemicarbazide derivatives.

Adenylylation of Gyrase and Topo IV by FicT Toxins Disrupts Bacterial DNA Topology.

Toxin-antitoxin (TA) modules are ubiquitous molecular switches controlling bacterial growth via the release of toxins that inhibit cell proliferation. Most of these toxins interfere with protein translation, but a growing variety of other mechanisms hints at a diversity that is not yet fully appreciated. Here, we characterize a group of FIC domain proteins as toxins of the conserved and abundant FicTA family of TA modules, and we reveal that they act by suspending control of cellular DNA topology. We show that FicTs are enzymes that adenylylate DNA gyrase and topoisomerase IV, the essential bacterial type IIA topoisomerases, at their ATP-binding site. This modification inactivates both targets by blocking their ATPase activity, and, consequently, causes reversible growth arrest due to the knotting, catenation, and relaxation of cellular DNA. Our results give insight into the regulation of DNA topology and highlight the remarkable plasticity of FIC domain proteins.

Inhibitors of the DNA-cleavage gate of bacterial type IIA topoisomerases

Type IIA topoisomerases resolve topological problems in DNA by making a double-stranded break in one DNA segment, passing another DNA duplex through this break, and then resealing the break. Drugs (such as the widely used fluoroquinolone antibacterials and anti-cancer compounds such as etoposide) that stabilize double-strandedly cleaved DNA complexes with type IIA topoisomerases are cytotoxic. In GlaxoSmithKline a new class of novel bacterial topoisomerase inhibitors (NBTIs) have been developed. A 2.1Å crystal structure of a complex of GSK299423 with DNA and S. aureus DNA gyrase showed how the NBTI inhibits the enzyme by interacting with both the DNA and the protein. A pocket occupied by the compound in the protein (at the dimer interface) is absent in the apo structure, while the pocket occupied by the compound in the DNA has been formed by the enzyme stretching and untwisting the DNA between the two active sites. The NBTI structure has trapped a pre-cleavage complex of the enzyme, before the four base-pair double stranded break has occurred, and the structure gives insights into the role of metal ions in the cleavage mechanism of type IIA topoisomerases. Stuctures suggest how relatively small movements at the active sites (for example an ~3Å movement of a magnesium ion) can cause the cleavage of phosphate ester bonds and are coupled to the large domain movements involved in the catalytic cycle of these conformationally flexible enzymes. The binding site for the NBTI is close to but distinct from those for fluoroquinolones. Structures shows how the fluoroquinolone interacts with both the protein and the DNA by binding a non-catalytic magnesium ion and four associated waters. This provides a structural explanation for both fluoroquinolone resistance mutations and SAR (structure-activity relationships). Mechanistic implications of recent structural studies will be discussed.

Prospects for Developing New Antibacterials Targeting Bacterial Type IIA Topoisomerases

The modulation of DNA topology by DNA gyrase and topoisomerase IV, both of which are type IIA topoisomerases and found in most bacteria, is a function vital to DNA replication, repair and decatenation. Despite the potential for resistance development, DNA gyrase and/or topoisomerase IV have been proven to be and remain highly attractive targets in antibacterial drug discovery due to their potential for dual targeting. The search for new GyrA and/or ParC inhibitors that can overcome the increasing spread of multidrug-resistant bacteria has been successfully focused in the last decades on the modification of the known fluoroquinolone scaffold as primarily guided by ligand-based design via classical structure-activity relationship studies and the optimisation of physicochemical properties. This focus has resulted in several novel fluoroquinolones that have been introduced into clinical practice since 2000, and several of these new compounds are currently in different phases of clinical trials. Due to increasing resistance to fluoroquinolones, a significant part of DNA gyrase research has shifted to the discovery of new GyrB and/or ParE inhibitors, which are commonly identified through fragment-based design as well as virtual screening techniques and structure-based hit optimisation programs. This research often results in lead compounds with potent inhibitory activity and promising antibacterial activity profiles. Nevertheless, it is important to understand how different physicochemical properties (e.g., logD and total polar surface area) and different structural motifs influence the compounds' permeability to ensure the efficient discovery of potent, small-molecule antibacterials particularly against Gram-negative strains.

Tricyclic GyrB/ParE (TriBE) Inhibitors: A New Class of Broad-Spectrum Dual-Targeting Antibacterial Agents

Increasing resistance to every major class of antibiotics and a dearth of novel classes of antibacterial agents in development pipelines has created a dwindling reservoir of treatment options for serious bacterial infections. The bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV, are validated antibacterial drug targets with multiple prospective drug binding sites, including the catalytic site targeted by the fluoroquinolone antibiotics. However, growing resistance to fluoroquinolones, frequently mediated by mutations in the drug-binding site, is increasingly limiting the utility of this antibiotic class, prompting the search for other inhibitor classes that target different sites on the topoisomerase complexes. The highly conserved ATP-binding subunits of DNA gyrase (GyrB) and topoisomerase IV (ParE) have long been recognized as excellent candidates for the development of dual-targeting antibacterial agents with broad-spectrum potential. However, to date, no natural product or small molecule inhibitors targeting these sites have succeeded in the clinic, and no inhibitors of these enzymes have yet been reported with broad-spectrum antibacterial activity encompassing the majority of Gram-negative pathogens. Using structure-based drug design (SBDD), we have created a novel dual-targeting pyrimidoindole inhibitor series with exquisite potency against GyrB and ParE enzymes from a broad range of clinically important pathogens. Inhibitors from this series demonstrate potent, broad-spectrum antibacterial activity against Gram-positive and Gram-negative pathogens of clinical importance, including fluoroquinolone resistant and multidrug resistant strains. Lead compounds have been discovered with clinical potential; they are well tolerated in animals, and efficacious in Gram-negative infection models.

Non-quinolone Inhibitors of Bacterial Type IIA Topoisomerases: A Feat of Bioisosterism

Non-quinolone Inhibitors of Bacterial Type IIA Topoisomerases: A Feat of Bioisosterism

Mycobacterium tuberculosis DNA gyrase possesses two functional GyrA-boxes

In contrast with most bacteria which possess two typeII topoisomerases (topoisomerase IV and DNA gyrase), Mycobacterium tuberculosis possesses only one, DNA gyrase, which is functionally a hybrid enzyme. Functional differences between the two typeIIA topoisomerases are thought to be specified by a CTD (C-terminal DNA-binding domain), which controls DNA recognition. To explore the molecular mechanism responsible for the hybrid functions of the M. tuberculosis DNA gyrase, we conducted a series of sequence analyses and structural and biochemical experiments with the isolated GyrA CTD and the holoenzyme. Although the CTD displayed a global structure similar to that of bona fide GyrA and ParC paralogues, it harbours a second key motif similar in all respects to that of the conserved GyrA-box sequence motif. Biochemical assays showed that the GyrA-box is responsible for DNA supercoiling, whereas the second GyrA-box-l (GyrA-box-like motif) is responsible for the enhanced decatenation activity, suggesting that the mechanistic originality of M. tuberculosis DNA gyrase depends largely on the particular DNA path around the CTD allowed for by the presence of GyrA-box-l. The results of the present study also provide, through phylogenetic exploration of the entire Corynebacterineae suborder, a new and broader insight into the functional diversity of bacterial typeIIA topoisomerases.