Functional Analysis of the HD and ACT domains of the E. coli signal-transducing Uridylyltransferase/Uridylyl-removing enzyme (UTaselUR)
Ouillette, Ryan Matthew
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UTase/UR is a key component of the bicyclic cascade transduction pathway that regulates nitrogen assimilation in Escherichia coli via the protein glutamine synthetase (GS). This study examined the effects that specific site-directed mutations of highly conserved residues in the HD (phosphohydrolase) and ACT domains of the uridylyltransferase/ uridylyl-removing enzyme (UTase/UR) had on protein function. The effect mutations had on UTase/UR mutants was examined by means of a GS assay, in which UTase/UR behavior was extrapolated from the activity level of GS. An attempt was also made to purify one version of the mutant protein along with the wild-type. We discovered that mutations of these highly conserved domains suggest a loss or reduction of the uridylyl-removing activity of UTase/UR, but the protein maintained its uridylyl-transferase ability. Also, our d~ta suggest that the UTase/UR protein cannot be purified using a polyhistidine tag and Ni-NTA column approach due to possible problems with protein folding. Further investigations should be carried out to determine how the mutant UTase/UR enzyme loses its uridylyl-removing ability and what prevents it from being able to be purified using a His-tag.