Mutagenesis and Functional Characterization of the Four Domains of GlnD, a Bifunctional Nitrogen Sensor Protein

Abstract

GlnD is a bifunctional uridylyltransferase/uridylyl-removing enzyme (UTase/UR) and is believed to be the primary sensor of nitrogen status in the cell by sensing the level of glutamine in enteric bacteria. It plays an important role in nitrogen assimilation and metabolism by reversibly regulating the modification of P II protein; P II in turn regulates a variety of other proteins. GlnD appears to have four distinct domains: an N-terminal nucleotidyltransferase (NT) domain; a central HD domain, named after conserved histidine and aspartate residues; and two C-terminal ACT domains, named after three of the allosterically regulated enzymes in which this domain is found. Here we report the functional analysis of these domains of GlnD from Escherichia coli and Rhodospirillum rubrum. We confirm the assignment of UTase activity to the NT domain and show that the UR activity is a property specifically of the HD domain: substitutions in this domain eliminated UR activity, and a truncated protein lacking the NT domain displayed UR activity. The deletion of C-terminal ACT domains had little effect on UR activity itself but eliminated the ability of glutamine to stimulate that activity, suggesting a role for glutamine sensing by these domains. The deletion of C-terminal ACT domains also dramatically decreased UTase activity under all conditions tested, but some of these effects are due to the competition of UTase activity with unregulated UR activity in these variants.

Nitrogen Assimilation in Escherichia coli: Putting Molecular Data into a Systems Perspective

“…The latter suggestion, however, is consistent with a recent analysis of the four distinct domains of E. coli GlnD (128). The site of GlnD homologous to the catalytic domains of nucleotidyl transferases, which is approximately 100 amino acids in length and located at the N terminus of GlnD (102,128), is essential for the uridylyl transferase activity (128). By implication, the remaining part of the protein (ϳ800 amino acids), or a subdomain of it, may be involved in the deuridylylation reaction.…”

“…The former suggestion is consistent with a kinetic analysis of UTase that indicated that both reactions catalyzed by UTase may occur at a single active center (127). The latter suggestion, however, is consistent with a recent analysis of the four distinct domains of E. coli GlnD (128). The site of GlnD homologous to the catalytic domains of nucleotidyl transferases, which is approximately 100 amino acids in length and located at the N terminus of GlnD (102,128), is essential for the uridylyl transferase activity (128).…”

“…By implication, the remaining part of the protein (ϳ800 amino acids), or a subdomain of it, may be involved in the deuridylylation reaction. Indeed, this remaining part of GlnD contains three other domains: one HD domain (128,129) and, further downstream, two ACT domains (128,130), in the middle and at the C terminus of GlnD, respectively (128,131). The HD domain and the ACT domain are named after the conserved His and Asp residues and after three enzymes in which this domain is found, respectively (128).…”

“…Indeed, this remaining part of GlnD contains three other domains: one HD domain (128,129) and, further downstream, two ACT domains (128,130), in the middle and at the C terminus of GlnD, respectively (128,131). The HD domain and the ACT domain are named after the conserved His and Asp residues and after three enzymes in which this domain is found, respectively (128). The HD and ACT domains harbor the uridylyl-removing activity and the binding site for glutamine, respectively, as determined by in vitro assays of GlnD variant proteins (128).…”

“…The HD domain and the ACT domain are named after the conserved His and Asp residues and after three enzymes in which this domain is found, respectively (128). The HD and ACT domains harbor the uridylyl-removing activity and the binding site for glutamine, respectively, as determined by in vitro assays of GlnD variant proteins (128). The uridylyl-removing activity is a hydrolytic reaction (reaction II) ( Table 5).…”