Regulation of the intersubunit ammonia tunnel in Mycobacterium tuberculosis glutamine-dependent NAD+ synthetase

2012 ◽  
Vol 443 (2) ◽  
pp. 417-426 ◽  
Author(s):  
Watchalee Chuenchor ◽  
Tzanko I. Doukov ◽  
Melissa Resto ◽  
Andrew Chang ◽  
Barbara Gerratana
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Active Site ◽  
Drug Target ◽  
Wild Type ◽  
Ammonia Transport ◽  
Site Variation ◽  
Ammonia Tunnel ◽  
Essential Enzyme ◽  
Nad Synthetase ◽  
Insight Into

Glutamine-dependent NAD+ synthetase is an essential enzyme and a validated drug target in Mycobacterium tuberculosis (mtuNadE). It catalyses the ATP-dependent formation of NAD+ from NaAD+ (nicotinic acid–adenine dinucleotide) at the synthetase active site and glutamine hydrolysis at the glutaminase active site. An ammonia tunnel 40 Å (1 Å=0.1 nm) long allows transfer of ammonia from one active site to the other. The enzyme displays stringent kinetic synergism; however, its regulatory mechanism is unclear. In the present paper, we report the structures of the inactive glutaminase C176A variant in an apo form and in three synthetase–ligand complexes with substrates (NaAD+/ATP), substrate analogue {NaAD+/AMP-CPP (adenosine 5′-[α,β-methylene]triphosphate)} and intermediate analogues (NaAD+/AMP/PPi), as well as the structure of wild-type mtuNadE in a product complex (NAD+/AMP/PPi/glutamate). This series of structures provides snapshots of the ammonia tunnel during the catalytic cycle supported also by kinetics and mutagenesis studies. Three major constriction sites are observed in the tunnel: (i) at the entrance near the glutaminase active site; (ii) in the middle of the tunnel; and (iii) at the end near the synthetase active site. Variation in the number and radius of the tunnel constrictions is apparent in the crystal structures and is related to ligand binding at the synthetase domain. These results provide new insight into the regulation of ammonia transport in the intermolecular tunnel of mtuNadE.

scholarly journals Establishing Virulence Associated Polyphosphate Kinase 2 as a drug target for Mycobacterium tuberculosis

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Mamta Singh ◽  
Prabhakar Tiwari ◽  
Garima Arora ◽  
Sakshi Agarwal ◽  
Saqib Kidwai ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutant Strain ◽  
Guinea Pigs ◽  
High Throughput Screening ◽  
Drug Target ◽  
Wild Type Strain ◽  
Wild Type ◽  
Inorganic Polyphosphate ◽  
Phosphate Donor ◽  
Microbial Stress

Abstract Inorganic polyphosphate (PolyP) plays an essential role in microbial stress adaptation, virulence and drug tolerance. The genome of Mycobacterium tuberculosis encodes for two polyphosphate kinases (PPK-1, Rv2984 and PPK-2, Rv3232c) and polyphosphatases (ppx-1, Rv0496 and ppx-2, Rv1026) for maintenance of intracellular PolyP levels. Microbial polyphosphate kinases constitute a molecular mechanism, whereby microorganisms utilize PolyP as phosphate donor for synthesis of ATP. In the present study we have constructed ppk-2 mutant strain of M. tuberculosis and demonstrate that PPK-2 enzyme contributes to its ability to cause disease in guinea pigs. We observed that ppk-2 mutant strain infected guinea pigs had significantly reduced bacterial loads and tissue pathology in comparison to wild type infected guinea pigs at later stages of infection. We also report that in comparison to the wild type strain, ppk-2 mutant strain was more tolerant to isoniazid and impaired for survival in THP-1 macrophages. In the present study we have standardized a luciferase based assay system to identify chemical scaffolds that are non-cytotoxic and inhibit M. tuberculosis PPK-2 enzyme. To the best of our knowledge this is the first study demonstrating feasibility of high throughput screening to obtain small molecule PPK-2 inhibitors.

scholarly journals d-Alanine–d-alanine ligase as a model for the activation of ATP-grasp enzymes by monovalent cations

2020 ◽  
Vol 295 (23) ◽  
pp. 7894-7904
Author(s):  
Jordan L. Pederick ◽  
Andrew P. Thompson ◽  
Stephen G. Bell ◽  
John B. Bruning
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Drug Target ◽  
Catalytic Mechanism ◽  
Thermus Thermophilus ◽  
Monovalent Cation ◽  
Antibiotic Drug ◽  
Domains Of Life ◽  
Alanine Dipeptide ◽  
Insight Into

The ATP-grasp superfamily of enzymes shares an atypical nucleotide-binding site known as the ATP-grasp fold. These enzymes are involved in many biological pathways in all domains of life. One ATP-grasp enzyme, d-alanine–d-alanine ligase (Ddl), catalyzes ATP-dependent formation of the d-alanyl–d-alanine dipeptide essential for bacterial cell wall biosynthesis and is therefore an important antibiotic drug target. Ddl is activated by the monovalent cation (MVC) K+, but despite its clinical relevance and decades of research, how this activation occurs has not been elucidated. We demonstrate here that activating MVCs bind adjacent to the active site of Ddl from Thermus thermophilus and used a combined biochemical and structural approach to characterize MVC activation. We found that TtDdl is a type II MVC-activated enzyme, retaining activity in the absence of MVCs. However, the efficiency of TtDdl increased ∼20-fold in the presence of activating MVCs, and it was maximally activated by K+ and Rb+ ions. A strict dependence on ionic radius of the MVC was observed, with Li+ and Na+ providing little to no TtDdl activation. To understand the mechanism of MVC activation, we solved crystal structures of TtDdl representing distinct catalytic stages in complex with K+, Rb+, or Cs+. Comparison of these structures with apo TtDdl revealed no evident conformational change on MVC binding. Of note, the identified MVC binding site is structurally conserved within the ATP-grasp superfamily. We propose that MVCs activate Ddl by altering the charge distribution of its active site. These findings provide insight into the catalytic mechanism of ATP-grasp enzymes.

scholarly journals Chemical validation of Mycobacterium tuberculosis phosphopantetheine adenylyltransferase using fragment linking and CRISPR interference

2020 ◽  
Author(s):  
Jamal El Bakali ◽  
Michal Blaszczyk ◽  
Joanna C. Evans ◽  
Jennifer A. Boland ◽  
William J. McCarthy ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Crystal Structures ◽  
Active Site ◽  
Drug Target ◽  
Potential Drug Target ◽  
Biosynthesis Pathway ◽  
Potential Drug ◽  
Antimicrobial Drugs ◽  
X Ray ◽  
Crispr Interference

AbstractThe coenzyme A (CoA) biosynthesis pathway has attracted attention as a potential target for much-needed novel antimicrobial drugs, including for the treatment of tuberculosis (TB), the lethal disease caused by Mycobacterium tuberculosis (Mtb). Seeking to identify the first inhibitors of Mtb phosphopantetheine adenylyltransferase (MtbPPAT), the enzyme that catalyses the penultimate step in CoA biosynthesis, we performed a fragment screen. In doing so, we discovered three series of fragments that occupy distinct regions of the MtbPPAT active site, presenting a unique opportunity for fragment linking. Here we show how, guided by X-ray crystal structures, we could link weakly-binding fragments to produce an active site binder with a KD < 20 μM and on-target anti-Mtb activity, as demonstrated using CRISPR interference. This study represents a big step toward validating MtbPPAT as a potential drug target and designing a MtbPPAT-targeting anti-TB drug.Abstract Figure

scholarly journals Pyrazinoic Acid Inhibits a Bifunctional Enzyme in Mycobacterium tuberculosis

2017 ◽  
Vol 61 (7) ◽  
Author(s):  
Moses Njire ◽  
Na Wang ◽  
Bangxing Wang ◽  
Yaoju Tan ◽  
Xingshan Cai ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutant Allele ◽  
Stringent Response ◽  
Signal Transducer ◽  
Wild Type ◽  
Content Type ◽  
Catalytic Activities ◽  
Molecular Determinants ◽  
Insight Into ◽  
Pyrazinoic Acid

ABSTRACT Pyrazinamide (PZA), an indispensable component of modern tuberculosis treatment, acts as a key sterilizing drug. While the mechanism of activation of this prodrug into pyrazinoic acid (POA) by Mycobacterium tuberculosis has been extensively studied, not all molecular determinants that confer resistance to this mysterious drug have been identified. Here, we report how a new PZA resistance determinant, the Asp67Asn substitution in Rv2783, confers M. tuberculosis resistance to PZA. Expression of the mutant allele but not the wild-type allele in M. tuberculosis recapitulates the PZA resistance observed in clinical isolates. In addition to catalyzing the metabolism of RNA and single-stranded DNA, Rv2783 also metabolized ppGpp, an important signal transducer involved in the stringent response in bacteria. All catalytic activities of the wild-type Rv2783 but not the mutant were significantly inhibited by POA. These results, which indicate that Rv2783 is a target of PZA, provide new insight into the molecular mechanism of the sterilizing activity of this drug and a basis for improving the molecular diagnosis of PZA resistance and developing evolved PZA derivatives to enhance its antituberculosis activity.

A QM/MM Investigation of the Catalytic Mechanism of Acetylene Hydratase: Insights into Engineering a More Effective Enzyme

2020 ◽  
Author(s):  
wenyuan zhang ◽  
Eric A.C. Bushnell
Keyword(s):  
Active Site ◽  
Cluster Model ◽  
Reaction Rate ◽  
Catalytic Mechanism ◽  
Single Step ◽  
Wild Type ◽  
Step Process ◽  
Rate Determining Step ◽  
Binding Phase ◽  
Insight Into

In the present investigation, a QM/MM approach was used to better understand the effect of the second environmental shell of the active site on the catalytic conversion of acetylene to acetaldehyde by acetylene hydratase (AH). In addition, the effect of substituting W-coordinating sulfur atoms with selenium atoms was done to provide insight into the influence of the W-coordinating atoms on the catalytic reaction. From the results, it found that the presence of the second shell environment had a significant effect on the reaction. Specifically, in the absence of the MM second shell environment(i.e., QM-cluster model), the reaction rate-determining step is defined by the first proton transfer step. In contrast, for the QM/MM model, the rate-determining step is defined by the water attacking step. Moreover, with the presence of the MM second shell environment, a key intermediate found in the DFT-cluster investigation does not exist in the QM/MM investigation. Rather, what was a two-step process in the DFT-cluster study was calculated to occur in a single step for the QM/MM study. Regarding the sulfur to selenium substitutions, it was found that Gibbs energy for the acetylene binding phase was significantly affected. Notably, the trans-position selenium made the binding of acetylene 65.6 kJ mol-1 less endergonic. Moreover, the overall reaction became 38.2 kJ mol-1 less endergonic compared to the wild type (WT) AH model. Thus, the substitution of key W-coordinating sulfur atoms with selenium atoms may offer a means to enhance the catalytic mechanism of AH considerably.

scholarly journals Protection of Mycobacterium tuberculosis from Reactive Oxygen Species Conferred by the mel2 Locus Impacts Persistence and Dissemination

2009 ◽  
Vol 77 (6) ◽  
pp. 2557-2567 ◽  
Author(s):  
Suat L. G. Cirillo ◽  
Selvakumar Subbian ◽  
Bing Chen ◽  
Torin R. Weisbrod ◽  
William R. Jacobs ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Mycobacterium Tuberculosis ◽  
Bacterial Species ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Wild Type ◽  
Primary Mechanism ◽  
Insight Into ◽  
Increased Susceptibility

ABSTRACT Persistence of Mycobacterium tuberculosis in humans represents a major roadblock to elimination of tuberculosis. We describe identification of a locus in M. tuberculosis, mel2, that displays similarity to bacterial bioluminescent loci and plays an important role during persistence in mice. We constructed a deletion of the mel2 locus and found that the mutant displays increased susceptibility to reactive oxygen species (ROS). Upon infection of mice by aerosol the mutant grows normally until the persistent stage, where it does not persist as well as wild type. Histopathological analyses show that infection with the mel2 mutant results in reduced pathology and both CFU and histopathology indicate that dissemination of the mel2 mutant to the spleen is delayed. These data along with growth in activated macrophages and infection of Phox−/− and iNOS−/− mice and bone marrow-derived macrophages suggest that the primary mechanism by which mel2 affects pathogenesis is through its ability to confer resistance to ROS. These studies provide the first insight into the mechanism of action for this novel class of genes that are related to bioluminescence genes. The role of mel2 in resistance to ROS is important for persistence and dissemination of M. tuberculosis and suggests that homologues in other bacterial species are likely to play a role in pathogenesis.

scholarly journals Docking Simulations and QM/MM Studies between Isoniazid Prodrug, Catalase-Peroxidase (KatG) and S315T Mutant fromMycobacterium tuberculosis

2007 ◽  
Vol 8 (2) ◽  
pp. 113-124 ◽  
Author(s):  
E. F. F. da Cunha ◽  
T. C. Ramalho ◽  
R. B. de Alencastro ◽  
E. R. Maia
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Active Site ◽  
Isonicotinic Acid ◽  
Theoretical Calculations ◽  
Binding Pattern ◽  
Wild Type ◽  
Docking Simulations ◽  
Catalase Peroxidase ◽  
Experimental Structure

Isoniazid (INH), an antibiotic used to treat tuberculosis (TB), is a prodrug requiring activation by theMycobacterium tuberculosisKatG (mtKatG). In the present work, theoretical calculations were carried out to locate the most energetically-favorable INH–KatG interaction modes using the experimental structure of a wild type and mutantmtKatG active site. The S315T mutation significantly affects the ability of the enzyme to convert INH to isonicotinic acidin vitro. The results showed that significant changes occur in the INH binding pattern when serine is replaced by threonine.

scholarly journals Evolving the naturally compromised chorismate mutase from Mycobacterium tuberculosis to top performance

2020 ◽  
Vol 295 (51) ◽  
pp. 17514-17534
Author(s):  
Jūrate˙ Fahrig-Kamarauskait≑ ◽  
Kathrin Würth-Roderer ◽  
Helen V. Thorbjørnsrud ◽  
Susanne Mailand ◽  
Ute Krengel ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Mycobacterium Tuberculosis ◽  
High Activity ◽  
Active Site ◽  
Shikimate Pathway ◽  
Catalytic Efficiency ◽  
Chorismate Mutase ◽  
Full Potential ◽  
Evolutionary Trajectories ◽  
Essential Enzyme

Chorismate mutase (CM), an essential enzyme at the branch-point of the shikimate pathway, is required for the biosynthesis of phenylalanine and tyrosine in bacteria, archaea, plants, and fungi. MtCM, the CM from Mycobacterium tuberculosis, has less than 1% of the catalytic efficiency of a typical natural CM and requires complex formation with 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase for high activity. To explore the full potential of MtCM for catalyzing its native reaction, we applied diverse iterative cycles of mutagenesis and selection, thereby raising kcat/Km 270-fold to 5 × 105m−1s−1, which is even higher than for the complex. Moreover, the evolutionarily optimized autonomous MtCM, which had 11 of its 90 amino acids exchanged, was stabilized compared with its progenitor, as indicated by a 9 °C increase in melting temperature. The 1.5 Å crystal structure of the top-evolved MtCM variant reveals the molecular underpinnings of this activity boost. Some acquired residues (e.g. Pro52 and Asp55) are conserved in naturally efficient CMs, but most of them lie beyond the active site. Our evolutionary trajectories reached a plateau at the level of the best natural enzymes, suggesting that we have exhausted the potential of MtCM. Taken together, these findings show that the scaffold of MtCM, which naturally evolved for mediocrity to enable inter-enzyme allosteric regulation of the shikimate pathway, is inherently capable of high activity.

Insight into Factor IXa Selectivity and Reactivity Using Kunitz-Type Inhibitors: Importance of Factor IXa Residue K98.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1942-1942
Author(s):  
Pierre F. Neuenschwander ◽  
Stephen R. Williamson ◽  
Armen Nalian
Keyword(s):  
Active Site ◽  
Coagulation Factor ◽  
Heparin Binding ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Loop Region ◽  
Factor Ixa ◽  
Kunitz Type ◽  
Type Factor ◽  
Insight Into

Abstract Previous studies by us have shown that blood coagulation factor IXa is relatively resistant to inhibition by the Kunitz-type inhibitor bovine pancreatic trypsin inhibitor (BPTI; aprotinin), but that this resistance can be partially alleviated by the presence of low molecular weight heparin. In order to gain insight into potential mechansims of factor IXa selectivity and its modulation by heparin we undertook an examination of the reactivity of factor IXa with several other Kunitz-type inhibitors: the Kunitz-type inhibitor domain of protease nexin-2 (PN2KPI) and the first two Kunitz-type inhibitor domains of tissue factor pathway inhibitor (TFPI K1 and TFPI K2). As expected, factor IXa exhibited remarkable specificity towards these highly-homologous inhibitors and expressed the following order of reactivity: PN2KPI > TFPI K1/K2 > BPTI. Surprisingly, the enhancing effect of heparin (enoxaparin) was limited to factor IXa reactivity with BPTI and was not observed with PN2KPI or TFPI inhibitory domains. This effect of heparin was not due to a simple template or bridging mechanism between factor IXa and BPTI since progressively smaller oligosaccharides (H14, H10, H6) retained function. We performed molecular modeling and molecular dynamic simulation studies that suggested that BPTI residue R39 may sterically clash with the 99 loop region of factor IXa (specifically residue K98). In contrast, the corresponding position in PN2KPI (G39) does not appear to clash with factor IXa residue K98 in modeling studies. In addition, the 99 loop region of factor IXa is in close proximity to the heparin binding site. Thus, based on these observations we hypothesized that factor IXa residue K98 could be restricting occupation of the factor IXa active site, and that this steric hindrance could be partly alleviated by heparin binding. We therefore examined a mutant of factor IXa in which residue K98 was mutated to A. The fIXK98A mutant was expressed as a zymogen in human 293 cells and purified by a combination of ion exchange and heparin affinity chromatography. The fIXK98A mutant exhibited identical procoagulant activity compared to wild-type factor IXa in a standard clotting assay. In addition, upon activation with RVV-X the fIXaK98A enzyme showed similar activity as wild-type factor IXa towards the small peptide substrate CBS 31.39. In sharp contrast, however, examination of the inhibition of fIXaK98A by BPTI showed a dramatic enhancement in inhibition compared to the wild-type enzyme: Ki = 20 μM in the absence of heparin and Ki = 10 μM in the presence of heparin for fIXaK98A; and Ki > 500 μM in the absence of heparin and Ki = 40 μM in the presence of heparin for wild-type factor IXa. We conclude from this that factor IX residue K98 limits access of specific molecules into the active site of factor IXa and protects it from inhibition by BPTI. It would seem that some or all of this interference is alleviated upon heparin binding to factor IXa. The smaller effect of heparin on the mutant enzyme (2-fold) compared to the wild-type enzyme (>10-fold) further supports the supposition that heparin binding to factor IXa may in part be facilitating movement of the 99 loop of factor IXa to enhance access to the active site. This may have implications in the selectivity and reactivity of factor IXa with other inhibitors as well as certain substrates and provides some important insight into factor IXa function.

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