The two opposing activities of adenylyl transferase reside in distinct homologous domains, with intramolecular signal transduction

Abstract

Adenylyl transferase (ATase) is the bifunctional effector enzyme in the nitrogen assimilation cascade that controls the activity of glutamine synthetase (GS) in Escherichia coli. This study addresses the question of whether the two antagonistic activities of ATase (adenylylation and deadenylylation) occur at the same or at different active sites. The 945 amino acid residue ATase has been truncated in two ways, so as to produce two homologous polypeptides corresponding to amino acids 1–423 (AT‐N) and 425–945 (AT‐C). We demonstrate that ATase has two active sites; AT‐N carries a deadenylylation activity and AT‐C carries an adenylylation activity. Glutamine activates the adenylylation reaction of the AT‐C domain, whereas α‐ketoglutarate activates the deadenylylation reaction catalysed by the AT‐N domain. With respect to the regulation by the nitrogen status monitor PII, however, the adenylylation domain appears to be dependent on the deadenylylation domain: the deadenylylation activity of AT‐N depends on PII‐UMP and is inhibited by PII. The adenylylation activity of AT‐C is independent of PII (or PII‐UMP), whereas in the intact enzyme PII is required for this activity. The implications of this intramolecular signal transduction for the prevention of futile cycling are discussed.

31P NMR and Genetic Analysis Establish hinT as the Only Escherchia coli Purine Nucleoside Phosphoramidase and as Essential for Growth under High Salt Conditions

“…The nucleotidylation post-translational modification has been observed for the regulation of the glutamine synthetase activity by two enzymes: uridylyl transferase and adenylyl transferase (39). Our data also indicate that both AMP-lysine and GMPlysine are excellent substrates for E. coli hinT and human Hint1 (Table I).…”

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

“…Indeed, mutants lacking glnE had neither adenylylation nor deadenylylation activity (92,93). The presence of these two activities within ATase was also confirmed by showing that highly purified ATase, expressed from the cloned E. coli glnE gene (94), contained both adenylylation and deadenylylation activity in vitro (95).…”

“…Although reactions I and II are functionally the opposite of one another, chemically, they are not the reversal of each other (95,96). The sum of the adenylylation reaction and the deadenylylation reaction (reaction III) equals the conversion of ATP and phosphate to ADP and pyrophosphate.…”

“…Free energy estimates of ATP and PP i hydrolysis under cytosolic conditions at pH 7.6 and 3 mM Mg 2ϩ are Ϫ50 and Ϫ20 kJ/mol, respectively (97), and therefore, free energy to an amount of 30 kJ/mol is dissipated by the sum of the two reactions (reaction III) (Table 4), but 50 kJ/mol if the product pyrophosphate would be hydrolyzed further to inorganic phosphate (reaction IV) ( Table 4). Here the ATase is special biochemically not only in that it is bifunctional (i.e., catalyzing two different reactions) but also in that its two activities are antagonistic: the enzyme is "ambiguous" (95). Its preference for either reaction might be toggled by the binding of allosteric effectors or covalent modification.…”

“…Each domain is specialized in one of the two reactions catalyzed by ATase. The N-terminal domain catalyzes the deadenylylation reaction, while the C-terminal domain catalyzes the adenylylation reaction (95,98,99). A central, regulatory "R" domain of some 200 amino acids is located between the N-and C-terminal domains (98,100).…”