Site directed mutagenesis of impC Our results suggest that impC d

Site directed mutagenesis of impC Our results suggest that impC does not have a critical role in inositol production and hence our inability to obtain an impC mutant may indicate that impC has a different or secondary function that prevents Blebbistatin concentration isolation of a mutant. For example, the enzyme might form part of an enzyme complex, and play a vital structural role in maintaining the integrity of that complex. Deletion of the gene would

then have both enzymatic and structural effects. An analogous situation was found with the E. coli SuhB protein; where phenotypes in suhB mutants were not related to IMPase activity, as a point mutation in the active ABT-888 purchase site did not produce the suppressing phenotype [40]. We therefore used the same approach to try to separate enzymatic activity from a structural role. A D93N change in E. coli SuhB and an equivalent D90N change in the human IMPase suppress activity [40, 46] (Figure 1B). Site-directed mutagenesis was used to introduce a corresponding mutation (D86N) in the M.

tuberculosis impC gene using the integrating plasmid pFM96 previously used for complementation. This plasmid (pFM123) was introduced into the SCO strain FAME7, and the resultant strain (FAME11) was streaked onto sucrose/inositol plates. DCO colonies were analysed, Chk inhibitor and, in contrast to the situation with pFM96, all were shown to be wild-type (n = 52). The fact that the functional impC gene could not be replaced

by this mutated gene, even in the presence of inositol (p < 0.01), shows that the mutation did inactivate enzymatic activity, and (assuming that the structure was not affected) that it is this enzymatic activity that is essential, rather than an additional structural role. Enzyme activities In order to gain a greater understanding of the function of these IMPases, we expressed impC as a recombinant protein. However, despite using different plasmid constructs and strategies, we were unable to obtain a soluble protein (not shown). As an alternative to directly assaying enzyme activity, we assayed IMPase activity in cell extracts of the mutant strains to obtain information about their relative contributions to inositol synthesis. We compared enzyme activities in whole cell Endonuclease extracts from the wild-type and mutant strains (Tables 3 and 4). Of the seven substrates tested, phosphate release as a result of adding the enzyme source was significantly higher than controls for fructose bisphosphate (FBP), the inositol phosphates, 5′ AMP and p-nitrophenyl-phosphate. Deletion of the impA, suhB, or cysQ genes made no significant difference to IMPase activity. The cysQ mutants had significantly less FBPase than the parent strain, (P < 0.05; t-test). However, the fructose FBPase activity in the H37Rv control for the cysQ mutants (Table 4) is significantly less than in H37Rv control used for impA and suhB mutants (P < 0.

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