scholarly journals Computational evidence of a new allosteric communication pathway between active sites and putative regulatory sites in the alanine racemase ofMycobacterium tuberculosis

2018 ◽  
Author(s):  
Jayanthy Jyothikumar ◽  
Sushil Chandani ◽  
Tangirala Ramakrishna
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Discovery ◽  
Side Effects ◽  
Active Site ◽  
Bacterial Infections ◽  
Active Sites ◽  
Pyridoxal Phosphate ◽  
Bacterial Species ◽  
Alanine Racemase ◽  
Docking Simulations

AbstractAlanine racemase, a popular drug target fromMycobacterium tuberculosis, catalyzes the biosynthesis of D-alanine, an essential component in bacterial cell walls. With the help of elastic network models of alanine racemase fromMycobacterium tuberculosis, we show that the mycobacterial enzyme fluctuates between two undiscovered states—a closed and an open state. A previous experimental screen identified several drug-like lead compounds against the mycobacterial alanine racemase, whose inhibitory mechanisms are not known. Docking simulations of the inhibitor leads onto the mycobacterial enzyme conformations obtained from the dynamics of the enzyme provide first clues to a putative regulatory role for two new pockets targeted by the leads. Further, our results implicate the movements of a short helix, behind the communication between the new pockets and the active site, indicating allosteric mechanisms for the inhibition. Based on our findings, we theorize that catalysis is feasible only in the open state. The putative regulatory pockets and the enzyme fluctuations are conserved across several alanine racemase homologs from diverse bacterial species, mostly pathogenic, pointing to a common regulatory mechanism important in drug discovery.Author summaryIn spite of the discovery of many inhibitors against the TB-causing pathogenMycobacterium tuberculosis, only a very few have reached the market as effective TB drugs. Most of the marketed TB drugs induce toxic side effects in patients, as they non-specifically target human cells in addition to pathogens. One such TB drug, D-cycloserine, targets pyridoxal phosphate moiety non-specifically regardless of whether it is present in the pathogen or the human host enzymes. D-cycloserine was developed to inactivate alanine racemase in TB causing pathogen. Alanine racemase is a bacterial enzyme essential in cell wall synthesis. Serious side effects caused by TB drugs like D-cycloserine, lead to patients’ non-compliance with treatment regimen, often causing fatal outcomes. Current drug discovery efforts focus on finding specific, non-toxic TB drugs. Through computational studies, we have identified new pockets on the mycobacterial alanine racemase and show that they can bind drug-like compounds. The location of these pockets away from the pyridoxal phosphate-containing active site, make them attractive target sites for novel, specific TB drugs. We demonstrate the presence of these pockets in alanine racemases from several pathogens and expect our findings to accelerate the discovery of non-toxic drugs against TB and other bacterial infections.

scholarly journals Rv1218c, an ABC Transporter of Mycobacterium tuberculosis with Implications in Drug Discovery

2010 ◽  
Vol 54 (12) ◽  
pp. 5167-5172 ◽  
Author(s):  
Meenakshi Balganesh ◽  
Sanjana Kuruppath ◽  
Nimi Marcel ◽  
Sreevalli Sharma ◽  
Anju Nair ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Discovery ◽  
Bacterial Infections ◽  
Fold Increase ◽  
Potential Candidate ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
Experimental Drugs ◽  
Physiological Relevance ◽  
In The Wild

ABSTRACT Efflux systems are important in determining the efficacy of antibiotics used in the treatment of bacterial infections. In the last decade much attention has been paid to studying the efflux pumps of mycobacteria. New classes of compounds are under investigation for development into potential candidate drugs for the treatment of tuberculosis. Quite often, these have poor bactericidal activities but exhibit excellent target (biochemical) inhibition. Microarray studies conducted in our laboratories for deciphering the mode of action of experimental drugs revealed the presence of putative ABC transporters. Among these transporters, Rv1218c was chosen for studying its physiological relevance in mediating efflux in Mycobacterium tuberculosis. A ΔRv1218c mutant of M. tuberculosis displayed a 4- to 8-fold increase in the inhibitory and bactericidal potency for different classes of compounds. The MICs and MBCs were reversed to wild-type values when the full-length Rv1218c gene was reintroduced into the ΔRv1218c mutant on a multicopy plasmid. Most of the compound classes had significantly better bactericidal activity in the ΔRv1218c mutant than in the wild-type H37Rv, suggesting the involvement of Rv1218c gene product in effluxing these compounds from M. tuberculosis. The implication of these findings on tuberculosis drug discovery is discussed.

scholarly journals Chlamydia trachomatis dapFEncodes a Bifunctional Enzyme Capable of Both D-Glutamate Racemase and Diaminopimelate Epimerase Activities

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
Author(s):  
George Liechti ◽  
Raghuveer Singh ◽  
Patricia L. Rossi ◽  
Miranda D. Gray ◽  
Nancy E. Adams ◽  
...  
Keyword(s):  
Amino Acid ◽  
Chlamydia Trachomatis ◽  
Bacterial Infections ◽  
Pyridoxal Phosphate ◽  
De Novo ◽  
Bacterial Species ◽  
Genetic Complementation ◽  
Enzyme Promiscuity ◽  
Peptidoglycan Synthesis ◽  
Glutamate Racemase

ABSTRACTPeptidoglycan is a sugar/amino acid polymer unique to bacteria and essential for division and cell shape maintenance. Thed-amino acids that make up its cross-linked stem peptides are not abundant in nature and must be synthesized by bacteriade novo.d-Glutamate is present at the second position of the pentapeptide stem and is strictly conserved in all bacterial species. In Gram-negative bacteria,d-glutamate is generated via the racemization ofl-glutamate by glutamate racemase (MurI).Chlamydia trachomatisis the leading cause of infectious blindness and sexually transmitted bacterial infections worldwide. While its genome encodes a majority of the enzymes involved in peptidoglycan synthesis, nomurIhomologue has ever been annotated. Recent studies have revealed the presence of peptidoglycan inC. trachomatisand confirmed that its pentapeptide includesd-glutamate. In this study, we show thatC. trachomatissynthesizesd-glutamate by utilizing a novel, bifunctional homologue of diaminopimelate epimerase (DapF). DapF catalyzes the final step in the synthesis ofmeso-diaminopimelate, another amino acid unique to peptidoglycan. Genetic complementation of anEscherichia coli murImutant demonstrated thatChlamydiaDapF can generated-glutamate. Biochemical analysis showed robust activity, but unlike canonical glutamate racemases, activity was dependent on the cofactor pyridoxal phosphate. Genetic complementation, enzymatic characterization, and bioinformatic analyses indicate that chlamydial DapF shares characteristics with other promiscuous/primordial enzymes, presenting a potential mechanism ford-glutamate synthesis not only inChlamydiabut also numerous other genera within thePlanctomycetes-Verrucomicrobiae-Chlamydiaesuperphylum that lack recognized glutamate racemases.IMPORTANCEHere we describe one of the last remaining “missing” steps in peptidoglycan synthesis in pathogenicChlamydiaspecies, the synthesis ofd-glutamate. We have determined that the diaminopimelate epimerase (DapF) encoded byChlamydia trachomatisis capable of carrying out both the epimerization of DAP and the pyridoxal phosphate-dependent racemization of glutamate. Enzyme promiscuity is thought to be the hallmark of early microbial life on this planet, and there is currently an active debate as to whether “moonlighting enzymes” represent primordial evolutionary relics or are a product of more recent reductionist evolutionary pressures. Given the large number ofChlamydiaspecies (as well as members of thePlanctomycetes-Verrucomicrobiae-Chlamydiaesuperphylum) that possess DapF but lack homologues of MurI, it is likely that DapF is a primordial isomerase that functions as both racemase and epimerase in these organisms, suggesting that specializedd-glutamate racemase enzymes never evolved in these microbes.

scholarly journals Identification of Enzymatic Active Sites with Unsupervised Language Modeling

2021 ◽  
Author(s):  
Loïc Kwate Dassi ◽  
Matteo Manica ◽  
Daniel Probst ◽  
Philippe Schwaller ◽  
Yves Gaetan Nana Teukam ◽  
...  
Keyword(s):  
Language Processing ◽  
Active Site ◽  
Active Sites ◽  
Functional Characterization ◽  
3D Structure ◽  
Ground Truth ◽  
Sequence Information ◽  
Docking Simulations ◽  
Domain Specific ◽  
Language Representation

The first decade of genome sequencing saw a surge in the characterization of proteins with unknown functionality. Even still, more than 20% of proteins in well-studied model animals have yet to be identified, making the discovery of their active site one of biology's greatest puzzle. Herein, we apply a Transformer architecture to a language representation of bio-catalyzed chemical reactions to learn the signal at the base of the substrate-active site atomic interactions. The language representation comprises a reaction simplified molecular-input line-entry system (SMILES) for substrate and products, complemented with amino acid (AA) sequence information for the enzyme. We demonstrate that by creating a custom tokenizer and a score based on attention values, we can capture the substrate-active site interaction signal and utilize it to determine the active site position in unknown protein sequences, unraveling complicated 3D interactions using just 1D representations. This approach exhibits remarkable results and can recover, with no supervision, 31.51% of the active site when considering co-crystallized substrate-enzyme structures as a ground-truth, vastly outperforming approaches based on sequence similarities only. Our findings are further corroborated by docking simulations on the 3D structure of few enzymes. This work confirms the unprecedented impact of natural language processing and more specifically of the Transformer architecture on domain-specific languages, paving the way to effective solutions for protein functional characterization and bio-catalysis engineering.

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.

Exploring the structure of glutamate racemase from Mycobacterium tuberculosis as a template for anti-mycobacterial drug discovery

2016 ◽  
Vol 473 (9) ◽  
pp. 1267-1280 ◽  
Author(s):  
Sinothai Poen ◽  
Yoshio Nakatani ◽  
Helen K. Opel-Reading ◽  
Moritz Lassé ◽  
Renwick C.J. Dobson ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Discovery ◽  
Drug Design ◽  
Crystal Structures ◽  
Active Site ◽  
Glutamate Racemase

The crystal structures of glutamate racemase (MurI) from M. tuberculosis and M. smegmatis have been determined and analysed as templates for drug discovery. They reveal a new dimeric architecture and active site features that could be valuable for drug design.

scholarly journals Identification of Enzymatic Active Sites with Unsupervised Language Modeling

2022 ◽  
Author(s):  
Loïc Kwate Dassi ◽  
Matteo Manica ◽  
Daniel Probst ◽  
Philippe Schwaller ◽  
Yves Gaetan Nana Teukam ◽  
...  
Keyword(s):  
Language Processing ◽  
Active Site ◽  
Active Sites ◽  
Functional Characterization ◽  
3D Structure ◽  
Ground Truth ◽  
Sequence Information ◽  
Docking Simulations ◽  
Domain Specific ◽  
Language Representation

The first decade of genome sequencing saw a surge in the characterization of proteins with unknown functionality. Even still, more than 20% of proteins in well-studied model animals have yet to be identified, making the discovery of their active site one of biology's greatest puzzle. Herein, we apply a Transformer architecture to a language representation of bio-catalyzed chemical reactions to learn the signal at the base of the substrate-active site atomic interactions. The language representation comprises a reaction simplified molecular-input line-entry system (SMILES) for substrate and products, complemented with amino acid (AA) sequence information for the enzyme. We demonstrate that by creating a custom tokenizer and a score based on attention values, we can capture the substrate-active site interaction signal and utilize it to determine the active site position in unknown protein sequences, unraveling complicated 3D interactions using just 1D representations. This approach exhibits remarkable results and can recover, with no supervision, 31.51% of the active site when considering co-crystallized substrate-enzyme structures as a ground-truth, vastly outperforming approaches based on sequence similarities only. Our findings are further corroborated by docking simulations on the 3D structure of few enzymes. This work confirms the unprecedented impact of natural language processing and more specifically of the Transformer architecture on domain-specific languages, paving the way to effective solutions for protein functional characterization and bio-catalysis engineering.

An overview of protein moonlighting in bacterial infection

2014 ◽  
Vol 42 (6) ◽  
pp. 1720-1727 ◽  
Author(s):  
Brian Henderson
Keyword(s):  
Virulence Factors ◽  
Bacterial Infections ◽  
Active Sites ◽  
Pathogenic Bacteria ◽  
Glyoxylate Cycle ◽  
Bacterial Species ◽  
Tca Cycle ◽  
Bacterial Protein ◽  
Protein Families ◽  
Moonlighting Proteins

We are rapidly returning to a world in which bacterial infections are a major health issue. Pathogenic bacteria are able to colonize and cause pathology due to the possession of virulence factors such as adhesins, invasins, evasins and toxins. These are generally specifically evolved proteins with selective actions. It is, therefore, surprising that most human bacterial pathogens employ moonlighting proteins as virulence factors. Currently, >90 bacterial species employ one or more moonlighting protein families to aid colonization and induce disease. These organisms employ 90 moonlighting bacterial protein families and these include enzymes of the glycolytic pathway, tricarboxylic acid (TCA) cycle, hexosemonophosphate shunt, glyoxylate cycle and a range of other metabolic enzymes, proteases, transporters and, also, molecular chaperones and protein-folding catalysts. These proteins have homologues in eukaryotes and only a proportion of the moonlighting proteins employed are solely bacterial in origin. Bacterial moonlighting proteins can be divided into those with single moonlighting functions and those with multiple additional biological actions. These proteins contribute significantly to the population of virulence factors employed by bacteria and some are obvious therapeutic targets. Where examined, bacterial moonlighting proteins bind to target ligands with high affinity. A major puzzle is the evolutionary mechanism(s) responsible for bacterial protein moonlighting and a growing number of highly homologous bacterial moonlighting proteins exhibit widely different moonlighting actions, suggesting a lack in our understanding of the mechanism of evolution of protein active sites.

scholarly journals Breaking a Strong Amide Bond: Structure and Properties of Dimethylformamidase

2019 ◽  
Author(s):  
Chetan Kumar Arya ◽  
Swati Yadav ◽  
Jonathan Fine ◽  
Ana Casanal ◽  
Gaurav Chopra ◽  
...  
Keyword(s):  
Nitrogen Source ◽  
Active Site ◽  
Active Sites ◽  
Bacterial Species ◽  
Amide Bond ◽  
Dimethyl Formamide ◽  
Hydrolytic Cleavage ◽  
Oligomeric State ◽  
Carbon And Nitrogen ◽  
Mononuclear Iron

AbstractDimethylformamidase (DMFase) breaks down the human-made synthetic solvent N,N-dimethyl formamide(DMF) used extensively in industry(1). DMF is not known to exist in nature and was first synthesized in 1893. In spite of the recent origin of DMF, certain bacterial species such as Paracoccus, Pseudomonas, and Alcaligenes have evolved pathways to breakdown DMF and use them as carbon and nitrogen source for growth(2, 3). The work presented here provides a molecular basis for the ability of DMFase from Paracoccus to function in exacting conditions of high solvent concentrations, temperature and ionic strength to catalyze the hydrolysis of a stable amide bond. The structure reveals a multimeric complex of the α2β2 type or (α2β2)2 type. One of the three domains of the large subunit and the small subunit are hitherto undescribed folds and as yet of unknown evolutionary origin. The active site is made of a distinctive mononuclear iron that is coordinated by two tyrosine residues and a glutamic acid residue. The hydrolytic cleavage of the amide bond is catalyzed at the Fe3+ site with a proximal glutamate probably acting as the base. The change in the quaternary structure is salt dependent with high salt resulting in the larger oligomeric state. Kinetic characterization reveals an enzyme that shows cooperativity between subunits and the structure provides clues on the interconnection between the active sites.Significance StatementN,N-dimethyl formamide(DMF) is a commonly used industrial solvent that was first synthesized in 1893. The properties that make DMF a highly desired solvent also makes it a difficult compound to breakdown. Yet, certain bacteria have evolved to survive in environments polluted by DMF and have enzymes that breakdown DMF and use it as their carbon and nitrogen source. The molecular structure of the enzyme that breaks down the stable amide bond in these bacteria, reveals two new protein folds and a unique mononuclear iron active site. The work reported here provides the structural and biochemical framework to query the evolutionary origins of the protein, as well as in engineering this enzyme for use in bioremediation of a human made toxic solvent.

Picking pockets to fuel antimicrobial drug discovery

2007 ◽  
Vol 35 (5) ◽  
pp. 980-984 ◽  
Author(s):  
W.N. Hunter
Keyword(s):  
Oxidative Stress ◽  
Enzyme Activity ◽  
Drug Discovery ◽  
Active Site ◽  
Drug Target ◽  
Active Sites ◽  
Microbial Pathogens ◽  
Apicomplexan Parasites ◽  
Trypanosomatid Parasites ◽  
The Right

The inhibition of essential enzymes in microbial pathogens offers a route to treatment of infectious diseases. However, although the biology of the organism dictates a need for a particular enzyme activity, this does not necessarily mean that the enzyme is a good drug target. The chemistry of the active site (size, shape and properties) determines the likelihood of finding a molecule with the right properties to influence drug discovery. Discriminating between good and less-good targets is important. Studies on enzymes involved in the regulation of oxidative stress and pterin/folate metabolism of trypanosomatid parasites and isoprenoid precursor biosynthesis in bacteria and apicomplexan parasites illustrates a range of active sites representing those that are challenging with respect to the discovery of potent inhibitors, to others that provide more promising opportunities in drug discovery.

Acyl-Enzymes as Thrombolytic Agents in Dog Models of Venous Thrombosis and Pulmonary Embolism

1984 ◽  
Vol 51 (02) ◽  
pp. 248-253 ◽  
Author(s):  
R J Dupe ◽  
P D English ◽  
R A G Smith ◽  
J Green
Keyword(s):  
Pulmonary Embolism ◽  
Side Effects ◽  
Venous Thrombosis ◽  
Active Site ◽  
Acute Pulmonary Embolism ◽  
Quantitative Model ◽  
Beagle Dog ◽  
Thrombosis Model ◽  
Derivatives Of

SummaryA quantitative model of venous thrombosis in the beagle dog is described. The model was adapted to permit ageing of isolated experimental clots in vivo. A model of acute pulmonary embolism in this species is also described. In the venous thrombosis model, infusion of streptokinase (SK) or SK-activated human plasmin gave significant lysis but bolus doses of SK. plasmin complex were ineffective. Active site anisoylated derivatives of SK. plasminogen complex, SK-activated plasmin and activator-free plasmin were all active when given as bolus doses in both models. At lytic doses, the acyl-enzymes caused fewer side-effects attributable to plasminaemia than the corresponding unmodified enzymes.

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