ABCMdb

PMID: 11171040

Lemaitre N, Callebaut I, Frenois F, Jarlier V, Sougakoff W
Study of the structure-activity relationships for the pyrazinamidase (PncA) from Mycobacterium tuberculosis.
Biochem J. 2001 Feb 1;353(Pt 3):453-8., [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCC8 p.Ala146Val ABCC8 p.Pro69Leu Among them, three mutants (D8G, K96T and S104R) had virtually no activity ( 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). Login to comment 58 ABCC8 p.Ala146Val ABCC8 p.Pro69Leu All the mutants harboured a single amino acid substitution located either in the conserved (D8G, F13S, P69L, K96T, Y103S, S104R and A146V) or non-conserved (T61P and T167I) regions identified previously in the M. tuberculosis PncA protein [5]. Login to comment 66 ABCC8 p.Ala146Val ABCC8 p.Pro69Leu The second group contained five mutants, F13S, Y103S, A146V, T61P and P69L, which retained a low level Table 1 PZase activities of wild-type (WT) and PncA mutant proteins AS, active site, defined by the residues located within a 6 A/ sphere from the putative active Cys-138 found at the centre of the catalytic cleft (shown in Figure 2). Login to comment 67 ABCC8 p.Ala146Val ABCC8 p.Pro69Leu Strain Specific activity (units/mg of protein) Location of the mutated residue in the predicted PncA secondary structure PncA WT* 1.33 - PncA WT† 4.43 - K96T 0.002 β3 (AS) D8G 0.004 Loop connecting β1 and αC (AS) S104R 0.004 Loop connecting β3 and αD (AS) F13S 0.06 Loop connecting β1 and αC (AS) Y103S 0.11 Loop connecting β3 and αD (AS) A146V 0.17 αE T61P 0.21 Loop connecting β2 and β3 P69L 0.25 Loop connecting β2 and β3 T167I 1.51 αF * Reference strain H37Rv. Login to comment 109 ABCC8 p.Ala146Val ABCC8 p.Pro69Leu The four remaining mutants, T61P, P69L, A146V and T167I, were characterized by the replacement of amino acid residues more distant from Cys-138, but three of them were altered in positions associated with structural elements contributing to the active site, i.e. Ala-146 in the αE helix (Figure 2D) and Thr-61 and Pro-69 in the long loop connecting strands β2 and β3 and holding Trp-68 (Figures 1A and 1B). Login to comment 119 ABCC8 p.Ala146Val ABCC8 p.Pro69Leu The five mutants F13S, Y103S, A146V, T61P and P69L exhibited a significant decrease in PncA activity (see Table 1). Login to comment 129 ABCC8 p.Ala146Val ABCC8 p.Pro69Leu The moderate level of activity (0.1-0.2 unit\mg) displayed by the three remaining mutants T61P, P69L and A146V seems to be correlated, in the putative PncA structure, to the location of the corresponding substituted amino acids, which are relatively distant from the active cysteine. Login to comment 135 ABCC8 p.Pro69Leu In addition, the two mutants T61P and P69L are characterized by substitutions involving a proline, a residue that is known to produce bends in regular secondary structures. Login to comment 136 ABCC8 p.Pro69Leu Therefore, it can be hypothesized that the 5-20-fold decrease in activity observed in the mutants T61P and P69L results from a more global change in the conformation of the PncA protein. Login to comment