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.

The PII protein is Escherichia coli's cognate transducer of the nitrogen signal to the NRII (NtrB)͞NRI (NtrC) two-component system and to adenylyltransferase. Through these two routes, PII regulates both amount and activity of glutamine synthetase. GlnK is the recently discovered paralogue of PII, with a similar trimeric x-ray structure. Here we show that PII and GlnK form heterotrimers, in E. coli grown in nitrogen-poor medium. In vitro, fully uridylylated heterotrimers of the two proteins stimulated the deadenylylation activity of adenylyltransferase, albeit to a lower extent than homotrimeric PII-UMP. Fully uridylylated GlnK did not stimulate, or hardly stimulated, the deadenylylation activity. We propose that uridylylated PII͞GlnK heterotrimers fine-regulate the activation of glutamine synthetase. The PII͞GlnK couple is a first example of prokaryotic signal transducer that can form heterotrimers. Advantages of hetero-oligomer formation as molecular mechanism for fine-regulation of signal transduction are discussed.I n enteric bacteria, the PII protein occupies a pivotal position in the network regulating the activity of glutamine synthetase (GS), itself a key enzyme in the assimilation of ammonia. The nitrogen status of the cell, as sensed by uridylyltransferase (UTase), is transferred to PII by adjusting the degree of uridylylation of the latter. Native PII signals a nitrogen-rich status whereas PII-UMP flags a nitrogen-poor status. From PII, the signal is transferred to two targets, i.e., adenylyltransferase (ATase) and NRII (NtrB), involved in the covalent modification of GS and in the transcriptional regulation of the gene encoding GS, respectively (refs. 1 and 2; Fig. 1).Escherichia coli can also contain a PII-like protein: i.e., GlnK (3, 4). PII and GlnK are similar, both structurally and functionally. The two proteins are each composed of 112 amino acid residues that are 67% identical and have virtually the same trimeric crystal structure (5-7). Each protein has been purified as homotrimer (5, 8). Both PII and GlnK can be modified by UTase at tyrosine-51 (4, 9-12). Functionally, PII and GlnK can address the same targets; i.e., in vitro, they can both regulate the activities of ATase and NRII (4, 11). However, the in vivo response of either target to PII exceeds that to GlnK. This may in part be because of a difference in the effect of the small molecule 2-oxoglutarate on the two signal proteins (11, 13).One functional aspect differs drastically between PII and GlnK; i.e., the expression of the glnB gene (which encodes PII) is constitutive with respect to the intracellular nitrogen status (14) whereas the expression of the glnK gene is regulated by it (4, 15). There is little or no GlnK after growth in nitrogen-rich conditions. This report describes the identification of PII͞GlnK-heterotrimers in E. coli after growing the cells in nitrogen-poor media and also shows that in vitro generated heterotrimers have lower activity than PII homotrimers. It suggests that GlnK may moderate the regulation of ammoni...

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These data definitively showed that CD26 was a component of ZP-modified proteins in vivo. In addition, the data suggested that at least one other protein of approximately 110 kDa was modified by covalent adduct formation with ZAG.