Study of the structure-activity relationships for the pyrazinamidase (PncA) from Mycobacterium tuberculosis.
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 (< or =0.004 unit/mg), five (F13S, T61P, P69L, Y103S and A146V) retained a low level of activity (0.06-0.25 unit/mg) and one (T167L) exhibited a wild-type activity (1.51 units/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 A 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 alpha-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.
Lemaitre
N
N
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.
Callebaut
I
I
Frenois
F
F
Jarlier
V
V
Sougakoff
W
W
D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D017931:DNA Primers; D011994:Recombinant Proteins; D000581:Amidohydrolases; C022490:pyrazinamide deamidase