Hyperthermophilic asparaginase mutants with enhanced substrate affinity and antineoplastic activity: structural insights on their mechanism of action

Bansal, Sorav; Srivastava, Ankit; Mukherjee, Goutam; Pandey, Ramendra Pati; Verma, Anita; Mishra, Prashant; Kundu, Bishwajit

doi:10.1096/fj.11-191254

Cited by 70 publications

(68 citation statements)

References 64 publications

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“…For cloning the N‐ and C‐terminal domains separately, a previously developed PfA clone was used as template . Primer pairs 5′‐CT GCT AGC GTG AAA ATT CTT CTA ATT G‐3′ and 5′‐AG GGA TCC TTA GTT AAC CAC GAG ATC TTC TC‐3′ were used for PCR amplification of the DNA sequences corresponding to NPfA while primer pairs 5′‐TA GCT AGC GTC CTA GTT ATC AAA CTA ATC C‐3′ and 5′‐GGC GGG ATC CTA ATC TCT AAG CTC TCC‐3′ were used for CPfA.…”

Section: Methodsmentioning

confidence: 99%

N‐terminal domain of Pyrococcus furiosus l‐asparaginase functions as a non‐specific, stable, molecular chaperone

et al. 2013

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The enzyme L-asparaginase of Pyrococcus furiosus (PfA) functions as a dimer with each monomer consisting of distinct N-and C-terminal domains (NPfA and CPfA, respectively), connected by a linker. Here we present data to show that NPfA functions as a non-specific molecular chaperone. Independently expressed NPfA refolded spontaneously whereas CPfA formed insoluble aggregates. However, when mixed and refolded together, NPfA augmented CPfA to fold with~90% recovery. NPfA also protected a variety of substrate proteins from thermal and refolding-mediated aggregation as monitored by a reduction in light scattering. The co-appearance of substrate protein with NPfA in antibody pull-down assays as well as in eluted gel filtration peaks indicated direct protein-protein interaction. These interactions were hydrophobic in nature as determined by 8-anilino-1-naphthalene sulfonic acid fluorescence. NPfA inhibited polyglutamine-mediated amyloid formation and also facilitated disintegration of preformed amyloid fibrils of amyloid-b (1-42) as determined by reverse-phase HPLC-based sedimentation assay and thioflavin T binding assays, respectively. Dynamic light scattering experiments suggested that NPfA readily assembled into polydispersed oligomeric species. With no sequence similarity to a-crystallin or any known molecular chaperone, we present here NPfA as a novel molecular chaperone. Structured digital abstract• Ab amyloid 1-42 and Ab amyloid 1-42 bind by fluorescence technology (View interaction)• alpha-amylase and alpha-amylase bind by light scattering (View interaction)• Ab amyloid 1-42 and Ab amyloid 1-42 bind by transmission electron microscopy (View interaction)• NPfa binds to BCA II by anti tag coimmunoprecipitation (View interaction)• MSG and MSG bind by light scattering (View interaction)

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Section: Methodsmentioning

confidence: 99%

N‐terminal domain of Pyrococcus furiosus l‐asparaginase functions as a non‐specific, stable, molecular chaperone

et al. 2013

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“…l-asparaginase is synthesized at a slow rate in malignant cells as a result of their reduced capability to synthesize l-asparagine synthetase as compared to normal cells. Thus applying l-asparaginase to tumor cells can deplete its content of l-asparagine rendering them unable to synthesize protein as well as RNA and DNA and inducing apoptosis in these cells (Bansal et al 2012). l-asparaginase is used in food industry to prevent the formation of acrylamide (carcinogenic toxicant) (Krishnapura et al 2016) in food products manufactured at temperatures exceeding 100 °C.…”

Section: Introductionmentioning

confidence: 99%

Production, characterization and bioinformatics analysis of l-asparaginase from a new Stenotrophomonas maltophilia EMCC2297 soil isolate

et al. 2020

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An exhaustive screening program was applied for scoring a promising l-asparaginase producing-isolate. The recovered isolate was identified biochemically and molecularly and its l-asparaginase productivity was optimized experimentally and by Response Surface Methodology. The produced enzyme was characterized experimentally for its catalytic properties and by bioinformatics analysis for its immunogenicity. The promising l-asparaginase producing-isolate was selected from 722 recovered isolates and identified as Stenotrophomonas maltophilia and deposited at Microbiological Resources Centre (Cairo Mircen) under the code EMCC2297. This isolate produces both intracellular (type I) and extracellular (type II) l-asparaginases with about 4.7 fold higher extracellular l-asparaginase productivity. Bioinformatics analysis revealed clustering of Stenotrophomonas maltophilia l-asparaginase with those of Pseudomonas species and considerable closeness to the two commercially available l-asparaginases of E. coli and Erwinia chrysanthemi. Fourteen antigenic regions are predicted for Stenotrophomonas maltophilia l-asparaginase versus 16 and 18 antigenic regions for the Erwinia chrysanthemi and E. coli l-asparaginases. Type II l-asparaginase productivity of the test isolate reached 4.7 IU/ml/h and exhibited maximum activity with no metal ion requirement at 37 °C, pH 8.6, 40 mM asparagine concentration and could tolerate NaCl concentration up to 500 mM and retain residual activity of 55% at 70 °C after half an hour treatment period. Application both of random mutation by gamma irradiation and Response Surface Methodology that determined 38.11 °C, 6.89 pH, 19.85 h and 179.15 rpm as optimum process parameters could improve the isolate l-asparaginase productivity. Maximum production of about 8 IU/ml/h was obtained with 0.4% dextrose, 0.1% yeast extract and 10 mM magnesium sulphate. In conclusion l-asparaginase of the recovered Stenotrophomonas maltophilia EMCC2297 isolate has characters enabling it to be used for medical therapeutic application.

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“…Interestingly, in the presence of asparagine, the β‐hairpin guarding the active site underwent structural reorganization into a flexible loop (Figure D). The flexibility of loop was proposed to regulate the accessibility of substrate to the active site in various other asparaginases . Though our in silico analysis presents new reaction mechanism of substrate hydrolysis by MtA, attempts of crystallization are being undertaken to obtain further insights.…”

Section: Resultsmentioning

confidence: 99%

Identification and validation of l‐asparaginase as a potential metabolic target against Mycobacterium tuberculosis

Kataria

Singh

Kundu

2018

J of Cellular Biochemistry

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Multidrug-resistant Mycobacterium tuberculosis (Mtb) has emerged as a major health challenge, necessitating the search for new molecular targets. A secretory amidohydrolase, l-asparaginase of Mtb (MtA), originally implicated in nitrogen assimilation and neutralization of acidic microenvironment inside human alveolar macrophages, has been proposed as a crucial metabolic enzyme. To investigate whether this enzyme could serve as a potential drug target, it was studied for structural details and active site-specific inhibitors were tested on cultured Mycobacterial strain. The structural details of MtA obtained through comparative modeling and molecular dynamics simulations provided insights about the orchestration of an alternate reaction mechanism at the active site. This was contrary to the critical Tyr flipping mechanism reported in other asparaginases. We report the novel finding of Tyr to Val replacement in catalytic triad I along with the structural reorganization of a β-hairpin loop upon substrate binding in MtA active site. Further, 5 MtA-specific, active-site-based inhibitors were obtained by following a rigorous differential screening protocol. When tested on Mycobacterium culture, 3 of these, M3 (ZINC 4740895), M26 (ZINC 33535), and doxorubicin showed promising results with inhibitory concentrations (IC ) of 431, 100, and 56 µM, respectively. Based on our findings and considering stark differences with human asparaginase, we project MtA as a promising molecular target against which the selected inhibitors may be used to counteract Mtb infection effectively.

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Hyperthermophilic asparaginase mutants with enhanced substrate affinity and antineoplastic activity: structural insights on their mechanism of action

Cited by 70 publications

References 64 publications

N‐terminal domain of Pyrococcus furiosus l‐asparaginase functions as a non‐specific, stable, molecular chaperone

N‐terminal domain of Pyrococcus furiosus l‐asparaginase functions as a non‐specific, stable, molecular chaperone

Production, characterization and bioinformatics analysis of l-asparaginase from a new Stenotrophomonas maltophilia EMCC2297 soil isolate

Identification and validation of l‐asparaginase as a potential metabolic target against Mycobacterium tuberculosis

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