scholarly journals Active site conformational changes upon reaction intermediate biotinyl-5'-AMP binding in biotin protein ligase from Mycobacterium tuberculosis

2014 ◽  
Vol 23 (7) ◽  
pp. 932-939 ◽  
Author(s):  
Qingjun Ma ◽  
Yusuf Akhter ◽  
Matthias Wilmanns ◽  
Matthias T. Ehebauer
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Conformational Changes ◽  
Active Site ◽  
Reaction Intermediate ◽  
Biotin Protein Ligase

scholarly journals Potent Inhibitors of a Shikimate Pathway Enzyme from Mycobacterium tuberculosis

2011 ◽  
Vol 286 (18) ◽  
pp. 16197-16207 ◽  
Author(s):  
Sebastian Reichau ◽  
Wanting Jiao ◽  
Scott R. Walker ◽  
Richard D. Hutton ◽  
Edward N. Baker ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Conformational Changes ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Condensation Reaction ◽  
Shikimate Pathway ◽  
Enzyme Reaction ◽  
Multidrug Resistant ◽  
Active Site Residues ◽  
Site Water

Tuberculosis remains a serious global health threat, with the emergence of multidrug-resistant strains highlighting the urgent need for novel antituberculosis drugs. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first step of the shikimate pathway for the biosynthesis of aromatic compounds. This pathway has been shown to be essential in Mycobacterium tuberculosis, the pathogen responsible for tuberculosis. DAH7PS catalyzes a condensation reaction between P-enolpyruvate and erythrose 4-phosphate to give 3-deoxy-d-arabino-heptulosonate 7-phosphate. The enzyme reaction mechanism is proposed to include a tetrahedral intermediate, which is formed by attack of an active site water on the central carbon of P-enolpyruvate during the course of the reaction. Molecular modeling of this intermediate into the active site reported in this study shows a configurational preference consistent with water attack from the re face of P-enolpyruvate. Based on this model, we designed and synthesized an inhibitor of DAH7PS that mimics this reaction intermediate. Both enantiomers of this intermediate mimic were potent inhibitors of M. tuberculosis DAH7PS, with inhibitory constants in the nanomolar range. The crystal structure of the DAH7PS-inhibitor complex was solved to 2.35 Å. Both the position of the inhibitor and the conformational changes of active site residues observed in this structure correspond closely to the predictions from the intermediate modeling. This structure also identifies a water molecule that is located in the appropriate position to attack the re face of P-enolpyruvate during the course of the reaction, allowing the catalytic mechanism for this enzyme to be clearly defined.

scholarly journals Inhibition and reaction mechanism of Mycobacterium tuberculosis anthranilate phosphoribosyltransferase: A potential target for novel drug design

2021 ◽  
Author(s):  
◽  
Preeti Kundu
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Reaction Mechanism ◽  
Drug Design ◽  
Transition State ◽  
Conformational Changes ◽  
Active Site ◽  
Binding Sites ◽  
Titration Calorimetry ◽  
Inhibitor Binding ◽  
Novel Drug

<p>Tuberculosis (TB), which is estimated to affect 2 billion individuals worldwide, is an infection predominately caused by Mycobacterium tuberculosis(M. tuberculosis). Of particular concern is the increasing prevalence of TB, which is becoming resistant to the treatments currently available. Anthranilate phosphoribosyltransferase (AnPRT) catalyses the formation of N-(5’-phosphoribosyl)anthranilate (PRA) from 5-phospho-α-ribose-1-diphosphate (PRPP) and anthranilate and plays an important role in the synthesis of an essential amino acid in M.tuberculosis. A strain with a genetic knockout of the trpD gene, which encodes for the AnPRT enzyme, was unable to cause disease, even in immune-deficient mice. Therefore, this enzyme is a potential drug target for the development of new treatments against TB and other infectious diseases. This research explores the synthesis of different substrates and potential transition state analogues in order to understand catalysis and inhibition of AnPRT enzymes to aid novel drug design. The first part of this study utilises “bianthranilate-like” phosphonate inhibitors that display effective inhibition of the AnPRT enzyme, with the lowest Ki value being 1.3 μM. It was found strong enzymatic inhibition increases with an increased length of the phosphonate linker that occupies multiple anthranilate binding sites within the anthranilate binding channel of the enzyme. Crystal studies of the enzyme in complex with the inhibitors were carried out in order to expose the binding interactions. The second part of this study investigates several new compounds that target the active site of M. tuberculosis AnPRT, based on a virtual screening approach. This approach identified a strong AnPRT inhibitor, which displays an apparent Ki value of 7.0 ± 0.4 μM with respect to both substrates. This study also exposed a conformational change at the active site of the enzyme that occurs on inhibitor binding. The observed conformational changes of the enzyme active site diminish the binding of the substrate PRPP. These pieces of information provide future inhibitor design strategies to aid the development of novel anti-TB agents that target the AnPRT enzyme. To elucidate the reaction mechanism of M. tuberculosis AnPRT, the third part of this study explores the substrate binding sites in detail. This study uses structural analysis, complemented by differential scanning fluorimetry (DSF) and isothermal titration calorimetry (ITC), to reveal detailed information of the substrate and inhibitor binding sites. The final part of this thesis presents the synthesis of various PRPP analogues and potential transition state mimics that were designed based on the likely reaction mechanism of the enzyme. This set of inhibitors includes a number of iminoribitol analogues that were developed to capture the geometry of the flattened ribose ring and include a nitrogen atom within the ring to mimic the positive charge characteristics that are expected in the oxocarbenium-ion-like transition state predicted for M. tuberculosis AnPRT. Additionally, we were able to solve the structure of M. tuberculosis AnPRT in complex with one of the potential transition state mimics, which was observed to bind at the active site of the enzyme. This structure provides new insight into the catalytic mechanism of the enzyme and creates an opportunity to develop more specific inhibitors against the M. tuberculosis AnPRT enzyme.</p>

scholarly journals Inhibition and reaction mechanism of Mycobacterium tuberculosis anthranilate phosphoribosyltransferase: A potential target for novel drug design

2021 ◽  
Author(s):  
◽  
Preeti Kundu
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Reaction Mechanism ◽  
Drug Design ◽  
Transition State ◽  
Conformational Changes ◽  
Active Site ◽  
Binding Sites ◽  
Titration Calorimetry ◽  
Inhibitor Binding ◽  
Novel Drug

<p>Tuberculosis (TB), which is estimated to affect 2 billion individuals worldwide, is an infection predominately caused by Mycobacterium tuberculosis(M. tuberculosis). Of particular concern is the increasing prevalence of TB, which is becoming resistant to the treatments currently available. Anthranilate phosphoribosyltransferase (AnPRT) catalyses the formation of N-(5’-phosphoribosyl)anthranilate (PRA) from 5-phospho-α-ribose-1-diphosphate (PRPP) and anthranilate and plays an important role in the synthesis of an essential amino acid in M.tuberculosis. A strain with a genetic knockout of the trpD gene, which encodes for the AnPRT enzyme, was unable to cause disease, even in immune-deficient mice. Therefore, this enzyme is a potential drug target for the development of new treatments against TB and other infectious diseases. This research explores the synthesis of different substrates and potential transition state analogues in order to understand catalysis and inhibition of AnPRT enzymes to aid novel drug design. The first part of this study utilises “bianthranilate-like” phosphonate inhibitors that display effective inhibition of the AnPRT enzyme, with the lowest Ki value being 1.3 μM. It was found strong enzymatic inhibition increases with an increased length of the phosphonate linker that occupies multiple anthranilate binding sites within the anthranilate binding channel of the enzyme. Crystal studies of the enzyme in complex with the inhibitors were carried out in order to expose the binding interactions. The second part of this study investigates several new compounds that target the active site of M. tuberculosis AnPRT, based on a virtual screening approach. This approach identified a strong AnPRT inhibitor, which displays an apparent Ki value of 7.0 ± 0.4 μM with respect to both substrates. This study also exposed a conformational change at the active site of the enzyme that occurs on inhibitor binding. The observed conformational changes of the enzyme active site diminish the binding of the substrate PRPP. These pieces of information provide future inhibitor design strategies to aid the development of novel anti-TB agents that target the AnPRT enzyme. To elucidate the reaction mechanism of M. tuberculosis AnPRT, the third part of this study explores the substrate binding sites in detail. This study uses structural analysis, complemented by differential scanning fluorimetry (DSF) and isothermal titration calorimetry (ITC), to reveal detailed information of the substrate and inhibitor binding sites. The final part of this thesis presents the synthesis of various PRPP analogues and potential transition state mimics that were designed based on the likely reaction mechanism of the enzyme. This set of inhibitors includes a number of iminoribitol analogues that were developed to capture the geometry of the flattened ribose ring and include a nitrogen atom within the ring to mimic the positive charge characteristics that are expected in the oxocarbenium-ion-like transition state predicted for M. tuberculosis AnPRT. Additionally, we were able to solve the structure of M. tuberculosis AnPRT in complex with one of the potential transition state mimics, which was observed to bind at the active site of the enzyme. This structure provides new insight into the catalytic mechanism of the enzyme and creates an opportunity to develop more specific inhibitors against the M. tuberculosis AnPRT enzyme.</p>

Structures of Mycobacterium tuberculosis Anthranilate Phosphoribosyltransferase Variants Reveal the Conformational Changes That Facilitate Delivery of the Substrate to the Active Site

Biochemistry ◽  
2015 ◽  
Vol 54 (39) ◽  
pp. 6082-6092 ◽  
Author(s):  
Tammie V. M. Cookson ◽  
Genevieve L. Evans ◽  
Alina Castell ◽  
Edward N. Baker ◽  
J. Shaun Lott ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Conformational Changes ◽  
Active Site

scholarly journals Active site lysine backbone undergoes conformational changes in the bacteriorhodopsin photocycle.

1994 ◽  
Vol 269 (10) ◽  
pp. 7387-7389
Author(s):  
H. Takei ◽  
Y. Gat ◽  
Z. Rothman ◽  
A. Lewis ◽  
M. Sheves
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Bacteriorhodopsin Photocycle

scholarly journals Structural evidence for a reaction intermediate mimic in the active site of a sulfite dehydrogenase

2020 ◽  
Vol 56 (68) ◽  
pp. 9850-9853
Author(s):  
Ahmed Djeghader ◽  
Melanie Rossotti ◽  
Saleh Abdulkarim ◽  
Frédéric Biaso ◽  
Guillaume Gerbaud ◽  
...  
Keyword(s):  
Active Site ◽  
Spectroscopic Evidence ◽  
Reaction Intermediate

We provide structural and spectroscopic evidence for a molybdenum–phosphate adduct mimicking a proposed reaction intermediate in the active site of a prokaryotic sulfite oxidizing enzyme.

Examination of a reaction intermediate in the active site of riboflavin synthase

2003 ◽  
Vol 31 (4) ◽  
pp. 278-287 ◽  
Author(s):  
Ya-Jun Zheng ◽  
Douglas B. Jordan ◽  
Der-Ing Liao
Keyword(s):  
Active Site ◽  
Reaction Intermediate
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