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Adenylyl cyclase catalyzes the synthesis of cyclic AMP (cAMP), which is of central importance in signal transduction, metabolism, and other cellular processes in both eukaryotes and prokaryotes. cAMP levels in the cell are primarily regulated by the modulation of adenylyl cyclase activity. Catabolic-enzyme synthesis in Escherichia coli is regulated by the cellular concentration of cAMP. Despite the vast literature on catabolite repression and the glucose effect that has accumulated over the past couple of decades (12, 14, 29), the precise molecular mechanism for glucose inhibition of cAMP synthesis in E. coli remains unclear. Adenylyl cyclase and the phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS) proteins have been implicated in catabolite repression. Energized by PEP and successively mediated by the PTS proteins, enzyme I, HPr, and enzyme(s) IIABC (25), PTS sugars are translocated into the cell as sugar phosphates. This results in the conversion of the PTS proteins from a phosphorylated state to a dephosphorylated state. Inhibition of adenylyl cyclase activity by the PTS sugars has long been known to be due to this event (15). Although genetic evidence suggests that the PTS-catalyzed phosphorylation of IIA^Glc is involved in the activation of adenylyl cyclase (5, 24), no direct biochemical evidence is yet available to support this model. E. coli IIA^Glc has two histidines, H75 and H90, in close proximity in the catalytic site (30). Histidine 90 is the target for phosphorylation by the phosphohistidine-containing phosphocarrier protein (HPr~P) (4). Histidine 75 is conserved throughout the known proteins IIA^Glc of bacterial systems (17). Histidine 75 is required for phosphorylated protein IIA^Glc (IIA^Glc~P) to act as a phosphate donor to protein IIBC^Glc and glucose (18). Based on these biochemical properties and the atomic structures of the IIA^Glc family members (10, 30), IIA^Glc catalytic-site mutants were created to define the role of IIA^Glc phosphorylation in the regulation of adenylyl cyclase activity. The results presented here demonstrate that IIA^Glc~P is an activator of adenylyl cyclase while IIA^Glc does not affect the basal enzyme activity.

DNA manipulations. Digestion of DNA with restriction enzymes was performed according to the manufacturers' recommendations. DNA fragments were separated by electrophoresis on SeaKem GTG agarose or NuSieve GTG agarose, and bands were excised and melted at 65°C in an equal volume of 10 mM Tris-HCl (pH 8.0)-1 mM EDTA. DNA fragments were purified by phenol extraction and ethanol precipitation. Ligation of DNA fragments was performed as described elsewhere (26). Strain C600 lambda lysogen was used as the host for transformation with the ligation mixtures and for isolation of recombinant plasmids. Competent cells of the E. coli strains used here were prepared by the Hanahan method (6).

Cloning of the crr gene into pACYC184 under the control of P_tet. In order to express protein IIA^Glc at levels as close to the physiological level as possible for studies on the regulation of adenylyl cyclase in the toluene-treated cells, the crr gene was cloned into low-copy-number plasmid pACYC184 (3) under the control of P_tet. The HindIII-SalI fragment of pACYC184 encompassing the tet promoter region and part of the structural gene was cloned into M13mp18. The three bases (5'TGT3') 5' to the tet initiation codon ATG were mutated to 5'CAT3' by site-directed mutagenesis (28) to create the NdeI restriction recognition sequence. The HindIII-SalI fragment with the NdeI recognition sequence was amplified by PCR with the M13 forward and reverse primers. The amplified DNA was restricted with HindIII and SalI and cloned back into pACYC184 lacking the same fragment. The NdeI-SalI fragment containing the crr gene was cloned into the newly created NdeI site and the SalI site of pACYC184 such that crr expression would be under the control of P_tet.

Cloning of the crr gene into the pRE expression vector and purification of protein IIA^Glc and the H75Q and H75E mutants. The wild-type and mutant crr structural gene(s) was cloned as NdeI-SalI fragments into the respective sites in the pRE1 expression vector (20). A recombinant containing the wild-type crr gene was introduced into E. coli MZ1 (cI857) (31). Expression and purification of wild-type protein IIA^Glc were accomplished as described previously (21). The mutant proteins were similarly purified after expression in the crr deletion strain TP2865 transformed with plasmid pRK248 carrying the temperature-sensitive repressor (1).

Other methods. The details of the adenylyl cyclase assay have been described elsewhere (7). Synthesis of [³²P]PEP was accomplished as described elsewhere (22). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8), and labeled proteins were detected by autoradiography. Protein was estimated by the Lowry method (11). Oligonucleotides for mutagenesis were synthesized by the phosphoramidite method on an Applied Biosystems 380B DNA synthesizer. DNA sequence was determined, using Sequenase, by the dideoxy method of Sanger et al. (27) as modified by Biggin et al. (2).

Site-directed mutagenesis of histidines in protein IIA^Glc. The single-stranded DNA containing the crr gene in M13mp18 with an NdeI site at the initiation codon (21) was the starting material for site-directed mutagenesis of histidines 75 and 90 to glutamine and glutamate at each position. The three-dimensional structure of protein IIA^Glc shows that the two histidine residues at positions 75 and 90 form the active site (Fig. 1A) (10, 30), H90 being the target for phosphorylation by HPr~P. It was demonstrated that the H75Q IIA^Glc mutant is phosphorylated by HPr~P at H90 but fails to serve as a phosphoryl donor in the subsequent PTS phosphoryl transfer reaction; thus, it is permanently phosphorylated in the presence of PEP and the general energy coupling proteins enzyme I and HPr (18). In contrast, the H90Q mutant cannot be phosphorylated at all. These mutant proteins allow the examination of whether the phosphorylated or nonphosphorylated IIA^Glc regulates adenylyl cyclase activity. In addition, the H75E and H90E mutant proteins were analyzed because the presence of a negative charge at the active site of this phosphocarrier protein may mimic the phosphorylated form of IIA^Glc.

View larger version (70K): [in this window] [in a new window]   FIG. 1.   The active sites of wild-type IIAGlc (A) and modeled H75E (B) and H90E (C) mutants. (A) The proximity of phosphorylatable H90 and the interactive H75 in the catalytic pocket of IIAGlc are shown. It is possible to replace H75 (B) and H90 (C) with glutamate without altering the rest of the structure and yet maintain electrostatic interactions between residues 75 and 90. The figure was produced based on the crystal structure of Bacillus subtilis IIAGlc (10), whose active site is very similar to that of the E. coli protein (30).

Regulation of adenylyl cyclase activity by the state of phosphorylation of IIA^Glc. Strain TP2865, which lacks the crr gene (9), was used to evaluate the ability of the wild-type and mutant IIA^Glcs to regulate adenylyl cyclase activity. The strain was transformed with plasmid pDIA100, which overproduces adenylyl cyclase about 10-fold (23). Strain TP2865/pDIA100 was transformed either with pACYC184 as a control or pACYC184 carrying the wild-type crr gene or with one of the five mutants, H75Q, H75E, H90Q, H90E, and H75E H90A. The results in Table 1 show that the activity of adenylyl cyclase, in the presence of potassium phosphate, is strongly stimulated by complementation of the crr deletion strain with wild-type IIA^Glc or the H75Q mutant. When the phosphorylation of the catalytic H90 residue is abolished by the H90Q or H90E mutation, adenylyl cyclase is not activated. This clearly demonstrates that the activation of adenylyl cyclase occurred by virtue of the phosphorylation at H90 of the wild-type and H75Q mutant of IIA^Glc, because the nonphosphorylatable H90Q mutant failed to activate the enzyme. The H90E mutant also failed to activate adenylyl cyclase. However, the ability of H75E IIA^Glc to activate adenylyl cyclase is intermediate compared to the abilities of the phosphorylated and dephosphorylated forms. This suggests either that the H90 moiety of the H75E IIA^Glc mutant is phosphorylated by HPr~P or that the H75E IIA^Glc mutant, with the negatively charged glutamate in the catalytic pocket, may mimic the phosphorylated form of the wild-type protein. To address these possibilities, a H75E H90A IIA^Glc double mutant was constructed in which phosphorylation at position 90 is prevented. Partial stimulation of adenylyl cyclase activity caused by H75E IIA^Glc was reversed by the double mutant. This result suggests that H75E IIA^Glc may be phosphorylated.

In vitro phosphorylation of IIA^Glc. It is interesting to note the major difference between the phosphorylation patterns of the protein IIA^Glc H75Q and H75E mutants (Fig. 2). While the glutamine mutant is phosphorylated by HPr~P (lane 3), the glutamate mutant does not appear to be phosphorylated by HPr~P (lane 4). The twofold activation of adenylyl cyclase by H75E IIA^Glc may be reconciled by the facts that H75 and H90 in the catalytic pocket are proximal and that the negatively charged glutamate in protein IIA^Glc H75E may function like phosphorylated H90 (Fig. 1B). Another plausible explanation for the activation of adenylyl cyclase by the protein IIA^Glc H75E mutant is that this mutant gets transiently phosphorylated at H90, resulting in partial activation of adenylyl cyclase, but is rapidly dephosphorylated by the repulsive forces of the adjacent negatively charged glutamate residue such that the mutant is not observed in a steady-state phosphorylated form (Fig. 2, lane 4). A twofold activation of adenylyl cyclase by the H75E mutant and the reversal of this activation by the H75E H90A double mutant favor the argument that a transient phosphorylation of H90 of the H75E mutant may occur. The inability of H90E IIA^Glc to activate adenylyl cyclase is not readily understood. Perhaps the carboxyl group of glutamate in the H90E mutant is buried compared to the phosphate group of H90 of the wild-type protein (Fig. 1C).

View larger version (53K): [in this window] [in a new window]   FIG. 2.   Phosphoacceptor ability of wild-type protein IIAGlc and mutants H75Q and H75E. The phosphorylation reaction mixture contained 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 1 mM dithiothreitol, 2 mM [32P]phosphoenolpyruvate, 3 µg of enzyme I, 6 µg of HPr, and 2 µg of IIAGlc as indicated. Lanes: 1, no IIAGlc; 2, wild-type IIAGlc; 3, H75Q IIAGlc; 4, H75E IIAGlc. Reaction mixtures were incubated at 37°C for 15 min, and proteins were separated on a sodium dodecyl sulfate-15% polyacrylamide gel. The 32P-labeled proteins were detected by autoradiography.

Relationship between adenylyl cyclase activation and phosphate pools. There is substantial evidence that the activation of adenylyl cyclase by potassium phosphate is mediated through the PTS proteins (16). Furthermore, there is a clear relationship between the stimulation of adenylyl cyclase by potassium phosphate and the inhibition of adenylyl cyclase by glucose. Glucose has no effect on adenylyl cyclase basal activity. Only when the basal activity is stimulated by potassium phosphate is inhibition of adenylyl cyclase activity by glucose observed. As expected, glucose or methyl--D-glucopyranoside inhibited adenylyl cyclase activity in the strain transformed with the plasmid containing wild-type crr (Table 1). Surprisingly, a very similar result was observed with the H75Q mutant: it has been clearly demonstrated that this mutant cannot be dephosphorylated by methyl--D-glucopyranoside when purified PTS proteins are used (18). It is conceivable that in an intact cell, glucose or methyl--D-glucopyranoside may be vectorially phosphorylated, albeit at a slow rate, by the mannose pathway. Indeed, the efficient fermentation of glucose as well as mannose by the H75Q mutant (data not shown) is consistent with this interpretation. Thus, in an intact cell with the H75Q mutation, dephosphorylation of HPr can occur, and it in turn can be phosphorylated in the reversible reaction by the phospho-H75Q mutant. Such a turnaround dephosphorylation of the H75Q mutant would result in the deactivation of adenylyl cyclase.

Speculation on the interaction of adenylyl cyclase with protein IIA^Glc. The crystal structure of protein IIA^Glc suggests that there may be no gross conformational change in the protein upon phosphorylation (10, 30). Thus, we suggest that the acquisition of a negative charge at H90~P is responsible for the interaction with adenylyl cyclase. Protein IIA^Glc~P and adenylyl cyclase may form a stable complex through charge interactions. Protein IIA^Glc~P is a positive effector and protein IIA^Glc is a negative effector of different enzymes. This concerted role of phosphorylated and dephosphorylated protein IIA^Glc has been demonstrated in the transport and catabolism of non-PTS sugars like lactose, glycerol, and maltose. While protein IIA^Glc acts as a negative effector of the transport systems of these non-PTS sugars by binding to the sugar permeases, protein IIA^Glc~P dissociates from the sugar permeases and does not interfere with the transport. Protein IIA^Glc~P is a positive effector of adenylyl cyclase and thereby increases levels of cAMP, which is required for the transcription of the catabolic operons of lactose, glycerol, and maltose should the cells encounter these sugars.