Structural and functional insights into an archaeal<scp>L</scp>-asparaginase obtained through the linker-less assembly of constituent domains. Corrigendum

Tomar, Rachana; Sharma, Pankaj; Srivastava, Ankit; Bansal, Sorav; Ashish, .; Kundu, Bishwajit

doi:10.1107/s205979831502361x

Cited by 10 publications

(2 citation statements)

References 55 publications

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“…53 Interestingly, we have recently reported a similar reorganization of the structured active site loop in another type I asparaginases (P. furiosus) that assists substrate entry and its conversion to product. 56 Therefore, LdAI appears to employ a distinct mechanism of action and warrants further investigation. Interestingly, a recently reported L-asparaginase from Mycobacterium tuberculosis (MyAnsA) shows a similar Val substitution for Tyr.…”

Section: Active Sitementioning

confidence: 99%

l-Asparaginase as a new molecular target against leishmaniasis: insights into the mechanism of action and structure-based inhibitor design

et al. 2015

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l-Asparaginases belong to a family of amidohydrolases that catalyze the conversion of l-asparagine into l-aspartic acid and ammonia. Although bacterial l-asparaginases have been used extensively as anti-leukemic agents, their possible role as potential drug targets for pathogenic organisms has not been explored. The presence of genes coding for putative l-asparaginase enzymes in the Leishmania donovani genome hinted towards the specific role of these enzymes in extending survival benefit to the organism. To investigate whether this enzyme can serve as a potential drug target against the Leishmania pathogen, we obtained structural models of one of the putative Leishmania l-asparaginase I (LdAI). Using an integrated computational approach involving molecular modelling, docking and molecular dynamics simulations, we found crucial differences between catalytic residues of LdAI as compared to bacterial l-asparaginases. The deviation from the canonical acid-base pair at triad I, along with the structural reorganization of a β-hairpin loop in the presence of a substrate, indicated an altogether new mechanism of action of the LdAI enzyme. Moreover, the finding of compositional and functional differences between LdAI and human asparaginase was used as a criterion to identify specific small molecule inhibitors. Through virtual screening of a library of 11 438 compounds, we report five compounds that showed favorable interactions with the active pocket of LdAI, without adversely affecting human asparaginase. One of these compounds when tested on cultured Leishmania promastigotes displayed a promising leishmanicidal effect. Overall, our work not only provides first hand mechanistic insights of LdAI but also proposes five strongly active compounds which may prove as effective anti-leishmaniasis molecules.

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Section: Active Sitementioning

confidence: 99%

l-Asparaginase as a new molecular target against leishmaniasis: insights into the mechanism of action and structure-based inhibitor design

et al. 2015

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“…Unfortunately, we could not make null mutant of this enzyme, so we opted to rational design of its specific inhibitors, for which we first resorted to complete characterization using various bioinformatics and biochemical approaches. In the absence of structural information, the SEC and SAXS analysis validated our earlier proposed dimeric model for LdAI and established that it is structurally similar to P. furiosus L-asparaginase (Bansal et al., 2010, Garg et al., 2015, Tomar et al., 2016) and earlier studied cytoplasmic L-asparaginases (Yun et al., 2007). We then screened out two potential inhibitors L1 and L2 based on structural information of the enzyme active site and their similarity to substrate.…”

Section: Discussionmentioning

confidence: 99%

L-Asparaginase of Leishmania donovani: Metabolic target and its role in Amphotericin B resistance

Singh¹,

Khan²,

Yadav³

et al. 2017

International Journal for Parasitology: Drugs and Drug Resistan

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Emergence of Amphotericin B (AmB) resistant Leishmania donovani has posed major therapeutic challenge against the parasite. Consequently, combination therapy aimed at multiple molecular targets, based on proteome wise network analysis has been recommended. In this regard we had earlier identified and proposed L-asparaginase of Leishmania donovani (LdAI) as a crucial metabolic target. Here we report that both LdAI overexpressing axenic amastigote and promastigote forms of L. donovani survives better when challenged with AmB as compared to wild type strain. Conversely, qRT-PCR analysis showed an upregulation of LdAI in both forms upon AmB treatment. Our data demonstrates the importance of LdAI in imparting immediate protective response to the parasite upon AmB treatment. In the absence of structural and functional information, we modeled LdAI and validated its solution structure through small angle X-ray scattering (SAXS) analysis. We identified its specific inhibitors through ligand and structure-based approach and characterized their effects on enzymatic properties (Km, Vmax, Kcat) of LdAI. We show that in presence of two of the inhibitors L1 and L2, the survival of L. donovani is compromised whereas overexpression of LdAI in these cells restores viability. Taken together, our results conclusively prove that LdAI is a crucial metabolic enzyme conferring early counter measure against AmB treatment by Leishmania.

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Domains of Pyrococcus furiosus l-asparaginase fold sequentially and assemble through strong intersubunit associative forces

et al. 2015

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Here, we report the folding and assembly of a Pyrococcus furiosus-derived protein, L-asparaginase (PfA). PfA functions as a homodimer, with each monomer made of distinct N- and C-terminal domains. The purified individual domains as well as single Trp mutant of each domain were subjected to chemical denaturation/renaturation and probed by combination of spectroscopic, chromatographic, quenching and scattering techniques. We found that the N-domain acts like a folding scaffold and assists the folding of remaining polypeptide. The domains displayed sequential folding with the N-domain having higher thermodynamic stability. We report that the extreme thermal stability of PfA is due to the presence of high intersubunit associative forces supported by extensive H-bonding and ionic interactions network. Our results proved that folding cooperativity in a thermophilic, multisubunit protein is dictated by concomitant folding and association of constituent domains directly into a native quaternary structure. This report gives an account of the factors responsible for folding and stability of a therapeutically and industrially important protein.

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Structural and functional insights into an archaealL-asparaginase obtained through the linker-less assembly of constituent domains. Corrigendum

Abstract: A correction is made to Fig. 7 in the article by Tomaret al.[(2014).Acta Cryst.D70, 3187–3197].

Cited by 10 publications

References 55 publications

l-Asparaginase as a new molecular target against leishmaniasis: insights into the mechanism of action and structure-based inhibitor design

l-Asparaginase as a new molecular target against leishmaniasis: insights into the mechanism of action and structure-based inhibitor design

L-Asparaginase of Leishmania donovani: Metabolic target and its role in Amphotericin B resistance

Domains of Pyrococcus furiosus l-asparaginase fold sequentially and assemble through strong intersubunit associative forces

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