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Structural stability and functional analysis of L-asparaginase from Pyrococcus furiosus

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Abstract

We report studies on an L-asparaginase from Pyrococcus furiosus, cloned and expressed in Escherichia coli and purified to homogeneity. Protein stability and enzyme kinetic parameters were determined. The enzyme was found to be thermostable, natively dimeric, and glutaminase-free, with optimum activity at pH 9.0. It showed a K m of 12 mM and a substrate inhibition profile above 20 mM L-asparagine. Urea could not induce unfolding and enzyme inactivation; however, with guanidine hydrochloride (GdnCl) a two-state unfolding pattern was observed. Reduced activity and an altered near-UV-CD signal for protein at low GdnCl concentration (1 M) suggested tertiary structural changes at the enzyme active site. A homology three-dimensional model was developed and the structural information was combined with activity and stability data to give functional clues about the asparaginase.

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Abbreviations

AMC:

7-amino-4-methylcoumarin

EcAII, ErA, PfA, PhA, TaqA, and TthA:

asparaginases from Escherichia coli, Erwinia chrysanthemi, Pyrococcus furiosus, P. horikoshii, Thermus aquaticus, and T. thermophilus

GdnCl:

guanidine hydrochloride

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Authors and Affiliations

  1. Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, 110016, India

    S. Bansal, D. Gnaneswari, P. Mishra & B. Kundu

  2. School of Biological Sciences, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, 110016, India

    B. Kundu

Authors
  1. S. Bansal
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  2. D. Gnaneswari
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  3. P. Mishra
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  4. B. Kundu
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Correspondence to B. Kundu.

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Published in Russian in Biokhimiya, 2010, Vol. 75, No. 3, pp. 457–464.

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Bansal, S., Gnaneswari, D., Mishra, P. et al. Structural stability and functional analysis of L-asparaginase from Pyrococcus furiosus . Biochemistry Moscow 75, 375–381 (2010). https://doi.org/10.1134/S0006297910030144

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