In an attempt to investigate the molecular basis of pyrazinamide hydrolysis by the PncA protein from Mycobacterium tuberculosis, we determined the pyrazinamidase activity of nine PncA mutants bearing a single amino acid substitution. Among them, three mutants (D8G, K96T and S104R) had virtually no activity (⩽ 0.004unit/mg), five (F13S, T61P, P69L, Y103S and A146V) retained a low level of activity (0.06–0.25unit/mg) and one (T167L) exhibited a wild-type activity (1.51units/mg). The possible structural effects of these substitutions were assessed by analysing a three-dimensional model of the PncA protein constructed on the basis of the crystal structure of the N-carbamoylsarcosine amidohydrolase (CSHase) from Arthrobacter sp., an amidohydrolase which was found by hydrophobic cluster analysis to be closely related to PncA. In the PncA model, five of the mutated residues, Asp-8, Phe-13, Lys-96, Tyr-103 and Ser-104, were located within a 6 Å sphere around the cysteine residue Cys-138, which could be the counterpart of the active cysteine residue Cys-177 found in the CSHase. Among the remaining mutated residues, Thr-61, Pro-69 and Ala-146 were found to be more distant from Cys-138 but were associated with structural elements contributing to the catalytic centre, whereas Thr-167 was situated in an α-helix located far from the putative active site. These data suggest that the decrease in pyrazinamidase activity observed in the PncA mutant proteins is well correlated with the structural modifications the mutations can cause in the environment of the putative active cysteine Cys-138.
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February 2001
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Research Article|
January 25 2001
Study of the structure–activity relationships for the pyrazinamidase (PncA) from Mycobacterium tuberculosis
Nadine LEMAITRE;
Nadine LEMAITRE
*Laboratoire de Recherche Moléculaire sur les Antibiotiques, Faculté de Médecine Pitié-Salpêtrière, Université Paris VI, 91 bvd de l'Hôpital, 75634 Paris Cedex 13, France
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Isabelle CALLEBAUT;
Isabelle CALLEBAUT
†Systèmes Moléculaires et Biologie Structurale, LMCP, CNRS UMR C7590, Universités Paris VI et Paris VII, case 115, 4 place Jussieu, 75252 Paris cedex 05, France
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Frédéric FRENOIS;
Frédéric FRENOIS
‡BioXtal, la Pépinière d'Entreprises de GIF, 2 route de la Noue, 91193 Gif sur Yvette cedex, France
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Vincent JARLIER;
Vincent JARLIER
*Laboratoire de Recherche Moléculaire sur les Antibiotiques, Faculté de Médecine Pitié-Salpêtrière, Université Paris VI, 91 bvd de l'Hôpital, 75634 Paris Cedex 13, France
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Wladimir SOUGAKOFF
Wladimir SOUGAKOFF
1
*Laboratoire de Recherche Moléculaire sur les Antibiotiques, Faculté de Médecine Pitié-Salpêtrière, Université Paris VI, 91 bvd de l'Hôpital, 75634 Paris Cedex 13, France
1To whom correspondence should be addressed (e-mail sougakof@lmcp.jussieu.fr).
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Publisher: Portland Press Ltd
Received:
August 17 2000
Revision Received:
September 18 2000
Accepted:
November 09 2000
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 2001
2001
Biochem J (2001) 353 (3): 453–458.
Article history
Received:
August 17 2000
Revision Received:
September 18 2000
Accepted:
November 09 2000
Citation
Nadine LEMAITRE, Isabelle CALLEBAUT, Frédéric FRENOIS, Vincent JARLIER, Wladimir SOUGAKOFF; Study of the structure–activity relationships for the pyrazinamidase (PncA) from Mycobacterium tuberculosis. Biochem J 1 February 2001; 353 (3): 453–458. doi: https://doi.org/10.1042/bj3530453
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