Figure 4
The putative catalytic triad of GatD/MurT. (a) Overlay of GatD/MurT with four glutamine amidotransferase fold-containing proteins obtained from a HHPRED26 search (white cartoon and sticks). Whereas most GATases possess a conserved catalytic triad consisting of cysteine, histidine and glutamate residues, the glutamate is replaced by a proline (GatD-P191) in the GatD sequence and the conserved histidine (GatD-H189) is oriented towards an aspartic acid (MurT-D349) in MurT. (b) Multiple sequence alignment of putative homologous GatD/MurT enzymes. Conservation is color-coded, with white indicating low conservation, grey medium, and dark blue indicating high conservation. Residues GatD-C94, GatD-H189 and MurT-D349 are highly conserved (red box), as well as their immediate surroundings. (c) Thin-layer chromatography analysis of an activity assay of catalytic triad mutants. Mutation of MurT-D349 to asparagine completely abolishes catalysis in vitro, similarly to GatD-C94 mutations to either serine or glycine.
