Activation of a Cyanobacterial Adenylate Cyclase, CyaC, by Autophosphorylation and a Subsequent Phosphotransfer Reaction*

The CyaC protein, a cyanobacterial adenylate cyclase, has a unique primary structure composed of the catalytic domain of adenylate cyclase and the conserved domains of bacterial two-component regulatory systems, one transmitter domain and two receiver domains. In the present work, CyaC was produced inEscherichia coli as a histidine-tagged recombinant protein and purified to homogeneity. CyaC showed ability to autophosphorylatein vitro with the γ-phosphate of [γ-32P]ATP. CyaC derivatives were constructed by site-directed mutagenesis in which the highly conserved phosphorylation sites in the transmitter domain (His572) and receiver domains (Asp60 or Asp895) were replaced by glutamine and alanine residues, respectively. After autophosphorylation of the CyaC derivatives, the chemical stabilities of the phosphoryl groups bound to the derivatives were determined. It was found that His572 is the initial phosphorylation site and that the phosphoryl group once bound to His572 is transferred to Asp895. The enzyme activities of the CyaC derivatives defective in His572 or Asp895 were considerably reduced. Asp895 is phosphorylated by acetyl [32P]phosphate, a small phosphoryl molecule, but Asp60 is not. Acetyl phosphate stimulates adenylate cyclase activity only when Asp895 is intact. These results suggest that the phosphorylation of Asp895 is essential for the activation of adenylate cyclase and that Asp60 functions differently from Asp895 in regulating the enzyme activity.

The CyaC protein, a cyanobacterial adenylate cyclase, has a unique primary structure composed of the catalytic domain of adenylate cyclase and the conserved domains of bacterial two-component regulatory systems, one transmitter domain and two receiver domains. In the present work, CyaC was produced in Escherichia coli as a histidine-tagged recombinant protein and purified to homogeneity. CyaC showed ability to autophosphorylate in vitro with the ␥-phosphate of [␥-32 P]ATP. CyaC derivatives were constructed by site-directed mutagenesis in which the highly conserved phosphorylation sites in the transmitter domain (His 572 ) and receiver domains (Asp 60 or Asp 895 ) were replaced by glutamine and alanine residues, respectively. After autophosphorylation of the CyaC derivatives, the chemical stabilities of the phosphoryl groups bound to the derivatives were determined. It was found that His 572 is the initial phosphorylation site and that the phosphoryl group once bound to His 572 is transferred to Asp 895 . The enzyme activities of the CyaC derivatives defective in His 572 or Asp 895 were considerably reduced. Asp 895 is phosphorylated by acetyl [ 32 P]phosphate, a small phosphoryl molecule, but Asp 60 is not. Acetyl phosphate stimulates adenylate cyclase activity only when Asp 895 is intact. These results suggest that the phosphorylation of Asp 895 is essential for the activation of adenylate cyclase and that Asp 60 functions differently from Asp 895 in regulating the enzyme activity.
Cyclic AMP (cAMP) is widely distributed from prokaryotes to eukaryotes as an important signaling molecule. In cyanobacteria, Gram-negative bacteria that are able to perform higher plant-type oxygen-evolving photosynthesis, it has been shown that cellular cAMP levels change in response to changes in environmental conditions such as light-dark, low pH-high pH, oxic-anoxic (1,2), and nitrogen replete-deplete (3). In a gliding cyanobacterium, Spirulina platensis, extracellular cAMP stimulates the activity of respiration and gliding movement (4). These results suggest that cAMP functions as a signaling molecule in cyanobacteria, although the regulatory mechanism of cellular cAMP is yet to be elucidated. cAMP is synthesized from ATP by an adenylate cyclase, and the activity of adenylate cyclase is regulated by various mechanisms. Mammalian transmembrane adenylate cyclases are regulated by the stimulatory and inhibitory heterotrimeric G proteins in response to the binding of chemical ligands to appropriate signal receptors (5). Ca 2ϩ and Ca 2ϩ /calmodulin also regulate mammalian transmembrane adenylate cyclases (5). The mammalian type V adenylate cyclase is phosphorylated by protein kinase C and protein kinase A, and phosphorylation regulates the activity of the enzyme (6,7). In budding yeast Saccharomyces cerevisiae, adenylate cyclase is activated by Ras, a small GTP-binding protein (8,9). In Escherichia coli, a phosphoenolpyruvate:carbohydrate phosphotransferase system regulates the adenylate cyclase activity (10,11).
Recently, a soluble adenylate cyclase has been isolated from mammalian testis (12). The catalytic domains of this soluble adenylate cyclase are more similar to those of cyanobacterial adenylate cyclases than they are to those of mammalian transmembrane adenylate cyclases (12).
Ten adenylate cyclase genes have been isolated from cyanobacteria (13)(14)(15)(16)(17). They have a fairly conserved catalytic domain near the C-terminal region and have characteristically different regulatory domains upstream of the catalytic domains. Among these genes, cyaC encodes a novel protein containing domains homologous to members of the bacterial twocomponent regulatory system, one transmitter domain and two receiver domains, in addition to the catalytic domain of adenylate cyclase (16). The CyaC protein seems to bind to the membrane of S. platensis cells, although it has no sufficient hydrophobic regions to pierce the membrane (16).
Bacterial two-component regulatory systems, which provide the dominant modes of signal transduction in bacteria for adaptation to environmental changes, consist of two families of signal transduction proteins, the sensory kinase family and the response regulator family (18,19). The sensory kinases undergo autophosphorylation at a histidine residue in their transmitter domain in response to environmental stimuli, and the phosphoryl group once bound to the histidine residue is transferred to an aspartate residue in the receiver domain of the cognate response regulator. In most cases, response regulators act as transcriptional factors and control the expression of target genes. CheB in E. coli and RegA in Dictyostelium discoideum exceptionally consist of a receiver domain and a catalytic domain of the enzyme, from methylesterase and phosphodiesterase, respectively. The enzymes are activated by phosphorylation of their receiver domains (20,21). Several two-component regulatory proteins, so-called "hybrid sensory kinases," contain both transmitter and receiver domains (22)(23)(24)(25). It can be said that CyaC has a novel structure consisting of both "a hybrid sensory kinase" and a catalytic domain in one molecule. Studies on the function of CyaC are important not only to understand regulation of adenylate cyclase in cyanobacteria, but also to address general issues about the regulation of enzyme activity by two-component regulatory systems.
In this study, we used recombinant proteins to investigate how CyaC is phosphorylated and whether the phosphorylation state controls the adenylate cyclase activity of CyaC.
Construction of CyaC Site-directed Mutants-pETA (16) contains the entire cyaC gene fused to a His tag sequence from pET28a vector (Novagen), which is placed under the control of the phage T7 promoter.
The CyaC D60A mutant was constructed with mismatch oligonucleotides and PCR 1 using ExTaq DNA polymerase (Takara). pETA was used for the template DNA. PCR was performed with the primers pD11 (5Ј position 159, GATCTGCTATACCGGAC, 3Ј position 175) and pD13 (5Ј position 281, CATCCGCTGTGCACTGATAATT, 3Ј position 260) or pD12 (5Ј position 260, AATTATCAGTGCACAGCGGATG, 3Ј position 281) and pD14 (5Ј position 467, ACCTTGCTGAACGATCG, 3Ј position 451), with mutated bases in boldface and the D60A substitution underlined. A new restriction site (ApaLI) was created at position 268 -273 without changing the amino acids. The ApaLI restriction enzyme was used in the screening for the mutagenized plasmid. The resulting PCR products were mixed, and PCR was repeated with primers pD11 and pD14. The PCR product was ligated into pCRII vector (Invitrogen) to make pCRD1. The DraI fragment of pCRD1 (220 base pairs) was ligated into pETA digested with DraI. The resulting construct, pETD1, contained the cyaC gene with the CyaC D60A mutation.
The CyaC H572Q mutant was constructed using methods similar to those for the D60A mutation. PCR was performed with primers pH1 (5Ј position 1149, ACGATTACCAATGCTATTC, 3Ј position 1167) and pH3 (5Ј position 1819, GTGCGGAATTCTTGGGAGAC, 3Ј position 1800) or pH2 (5Ј position 1800, GTCTCCCAAGAATTCCGCAC, 3Ј position 1819) and pH4 (5Ј position 2010, ACTCCACCAGGTCACAG, 3Ј position 1994), with mutated bases in boldface and the H572Q substitution underlined. A new restriction site (EcoRI) was created at position 1809 -1814 without changing the amino acids. The EcoRI restriction enzyme was used in the screening for the mutagenized plasmid. The resulting PCR products were mixed, and PCR was repeated with primers pH1 and pH4. The PCR product was ligated into pCRII vector to make pCRH. The MfeI-BsmI fragment of pCRH (770 base pairs) was ligated into pETA digested with MfeI and BsmI (partial digestion). The resulting construct, pETH, contained the cyaC gene with the CyaC H572Q mutation.
The CyaC D895A mutant was constructed using methods similar to those for the D60A mutation. PCR was performed with primers pD21 (5Ј position 2504, CGTAGAATTGGCTGATG, 3Ј position 2520) and pD23 (5Ј position 2788, GGCATCATTAATGCAGTAACA, 3Ј position 2768), or pD22 (5Ј position 2768, TGTTACTGCATTAATGATGCC, 3Ј position 2788) and pD24 (5Ј position 3268, ACAAACTTGTCAACAGTC, 3Ј position 3251), with mutated bases in boldface and the D895A substitution underlined. A new restriction site (AseI) was created at position 2777-2782 without changing the amino acids. The AseI restriction enzyme was used in the screening for the mutagenized plasmid. The resulting PCR products were mixed, and PCR was repeated with primers pD21 and pD24. The PCR product was ligated into pCRII vector to make pCRD2. The BsrGI-AvaI fragment of pCRD2 (670 base pairs) was ligated into pETA digested with BsrGI and AvaI (partial digestion). The resulting construct, pETD2, contained the cyaC gene with the CyaC D895A mutation.
The portions of pCRD1, pCRH, and pCRD2 derived from the PCR fragment were sequenced to ensure the existence of no other mutations.
The CyaC D60A,D895A double mutant was constructed using pETD1 and pETD2. Both pETD1 and pETD2 were digested with BamHI and BsrG I. The 2.5-kb pETD1 fragment was ligated into the 6.5-kb pETD2 fragment. The resulting construct, pETD1D2, contains the cyaC gene with the CyaC D60A,D895A double mutation.

Purification of Wild-type and Mutant His-CyaC Proteins-
The purification procedure was essentially the same as that described previously (16). The transformants, BL21(DE3)pLysS cells harboring one of the expression constructs of the CyaC derivatives, were grown at 25°C in Luria-Bertani medium (1.5 liters) supplemented with kanamycin (25 g ml Ϫ1 ) and chloramphenicol (30 g ml Ϫ1 ). Each recombinant cyaC gene was expressed in exponentially growing cells by adding 1 mM isopropyl-␤-D-thiogalactopyranoside. After 6 h, the cells were harvested by centrifugation, washed with 50 mM Tris-HCl (pH 7.5) buffer containing 150 mM NaCl, and resuspended in 50 ml of extraction buffer consisting of 50 mM Tris-HCl (pH 8.0), 10% (w/v) glycerol, 0.5 M NaCl, 0.05% (w/v) Tween 80, 5 mM imidazole, 1 mM PMSF, and 1 mg ml Ϫ1 lysozyme. The cell suspension was incubated on ice for 30 min and then sonicated at 4°C for 9 min (3 min ϫ 3) using a Kubota model 200M sonicator. The cell extract was centrifuged at 16,000 ϫ g for 10 min, and the supernatant was further centrifuged at 150,000 ϫ g for 40 min. The 150,000 ϫ g supernatant was loaded onto a HiTrap Chelating column (Amersham Pharmacia Biotech; 1.6 ϫ 2.5 cm) connected to a fast protein liquid chromatography system (Amersham Pharmacia Biotech) and eluted using step gradients of 5, 30, 60, and 200 mM imidazole in Buffer A (50 mM Tris-HCl (pH 8.0), 10% (w/v) glycerol, 0.5 M NaCl, 0.05% (w/v) Tween 80). The volume of eluent used in each step gradient was 32, 16, 13, and 8.5 ml, respectively. The 200 mM imidazole fraction was concentrated to 0.5 ml using Ultrafree-15 (Millipore). The concentrated eluate was loaded onto a Superose 6 column (Amersham Pharmacia Biotech; 1 ϫ 30 cm) and eluted with Buffer A containing 5 mM imidazole at a flow rate of 0.3 ml min Ϫ1 .
The phosphorylation assay using acetyl [ 32 P]phosphate was essentially the same as that described by Quon et al. (28). The synthesized acetyl [ 32 P]phosphate (7.5 l) was combined with each purified His-CyaC derivative in 7.5 l of 50 mM Tris-HCl (pH 8.0) buffer containing 2 mM DTT and 1 mM PMSF. The reactions were run at 30°C for 15 min and terminated by the addition of 5 l of 4 ϫ SDS-PAGE loading buffer. The samples were heated immediately at 50°C for 5 min and applied to an 8% SDS-PAGE gel. The proteins were subsequently blotted onto an Immobilon-P membrane (Millipore). The phosphorylated proteins were analyzed using a BAS1000 Imaging Analyzer (Fuji film).
Adenylate Cyclase Activity Assay-Unless otherwise stated, the assay mixture (final volume 0.2 ml) contained, besides the enzyme, 50 mM Tris-HCl (pH 8.0), 200 mM KCl, 0.1 mM ATP, 1 mM MgCl 2 , 2 mM DTT, and 1 mM PMSF. The reaction was run for 15 min at 30°C, terminated by the addition of 0.3 ml of 10% perchloric acid, and neutralized with 0.5 ml of 1.01 M KOH. The mixture was centrifuged at 200 ϫ g for 10 min, and the cAMP content in the supernatants was measured by an enzyme immunoassay system according to the manufacture's protocol (EIA system, Amersham Pharmacia Biotech).
Stability of Phosphate Links in Wild-type and Mutant His-CyaC Proteins-Wild-type and mutant His-CyaC proteins were phosphorylated and blotted onto an Immobilon-P membrane (Millipore) as described above. The blots were incubated in 1 M HCl, 3 M NaOH, or 50 mM Tris-HCl (pH 7.2) at 25°C. After incubation for 1 h, the membranes were washed briefly with distilled water, dried in air, and then exposed to an x-ray film.

RESULTS
Purification of His-CyaC-The CyaC protein has a multidomain structure consisting of the catalytic domain of adenylate cyclase and domains that are homologous to those of bacterial two-component regulatory systems. These domains exist in line from the N terminus: a receiver domain (R1), a transmitter domain, a receiver domain (R2), and a catalytic domain. The region between the receiver (R1) domain and the transmitter domain shows similarity to the ETR1 protein that has been identified as an ethylene receptor in plants (29,30).
Eight kinds of CyaC derivatives were constructed and each protein was fused with a histidine tag (Fig. 1A). The proteins were purified as described under "Experimental Procedures," and the purities were determined by SDS-PAGE (Fig. 1B). One band with a molecular mass of 140 kDa was observed in each lane.
In the previous work, we showed that the catalytic activity of the CyaC protein is stimulated by Mn 2ϩ and less extensively by Mg 2ϩ (16). The specific activities (Mg 2ϩ -or Mn 2ϩ -dependent adenylate cyclase activity) of the purified wild-type His-CyaC protein used in this study were 7.4 and 260 nmol of cAMP formed mg Ϫ1 ⅐min Ϫ1 , respectively. The specific activities are higher than those used in the previous work (16). This is due to improvements in the purification procedure as described under "Experimental Procedures." The molecular mass of His-CyaC was estimated to be 590 kDa by Superose 6 column chromatography, a kind of gel permeation column chromatography, suggesting that His-CyaC forms a homotetramer in E. coli.
Autophosphorylation of His-CyaC-Autophosphorylation of His-CyaC was assayed by incubating the proteins with [␣-32 P]ATP or [␥-32 P]ATP and Mg 2ϩ or Mn 2ϩ . Radiolabeling was obtained when His-CyaC was incubated with [␥-32 P]ATP but not with [␣-32 P]ATP (Fig. 2), indicating that the phosphorylation of His-CyaC is ␥-phosphate specific. Mg 2ϩ and Mn 2ϩ had similar effects on the autophosphorylation activity of His-CyaC (Fig. 2, lanes 2 and 4), although the adenylate cyclase activity of His-CyaC was stimulated by Mn 2ϩ (16). Mn 2ϩ might activate the catalytic activity of CyaC independently of the autophosphorylation activity.
Autophosphorylation of Mutant His-CyaC Proteins-The His 572 residue of CyaC corresponds to the highly conserved histidine residue found in sensory kinases of bacterial twocomponent regulatory systems. The Asp 60 and Asp 895 residues of CyaC correspond to a highly conserved aspartate residue among the response regulators of bacterial two-component regulatory systems (16,18,19).

Stability of the Phosphoryl Groups of the Phosphorylated Wild-type and Mutant
His-CyaC Proteins-Chemical stability assays have been performed to determine the class of phosphorylated amino acid. N-Phosphate bonds (phosphohistidine or phospholysine) are base-stable and acid-labile. Acyl phosphate bonds (phosphoaspartate or phosphoglutamate) are labile to both acid and base, and O-phosphate bonds (phosphoserine, phosphothreonine, or phosphotyrosine) are stable to acid (31,32). To compare the stability of phosphate links in the phosphorylated wild-type and mutant His-CyaC proteins, each phosphorylated product was subjected to SDS-PAGE and transferred to an Immobilon-P membrane. The membrane strips were treated with acid, base, or neutral buffer.
Adenylate Cyclase Activities of Wild-type and Mutant His-CyaC Proteins-The adenylate cyclase activities of His-CyaC derivatives were compared with that of His-CyaC(wild-type) in vitro (Fig. 5). The components in the reaction mixtures and the assay conditions were the same as those used for the autophosphorylation reaction except that the final volume of the reaction mixtures differed. The adenylate cyclase activities of His-CyaC(D60A), His-CyaC(H572Q), His-CyaC(D895A), and His-CyaC(D60A,D895A) were 75, 23, 21, and 11% that of His-CyaC(wild-type), respectively. The adenylate cyclase activity of His-CyaC was decreased considerably by replacement of the His 572 or Asp 895 residues with other amino acids.
Effect of Acetyl Phosphate on the Adenylate Cyclase Activities of His-CyaC Proteins-The adenylate cyclase activities of His-CyaC(H572Q), His-CyaC(H572Q,D60A), His-CyaC(H572Q, D895A), and His-CyaC(H572Q,D60A,D895A) were measured in the presence or absence of acetyl phosphate. Acetyl phosphate greatly activated the adenylate cyclase activities of His-CyaC(H572Q) and His-CyaC(H572Q,D60A) (Fig. 7), while His-CyaC(H572Q,D895A) and His-CyaC(H572Q,D60A,D895A) were largely unaffected. Thus, the presence of Asp 895 is essential for the activation of adenylate cyclase activity. Acetyl phosphate might activate the catalytic activity of the adenylate cyclase by phosphorylating Asp 895 in His-CyaC. DISCUSSION A cyanobacterial adenylate cyclase, CyaC, has been shown to have a unique structure consisting of one transmitter domain and two receiver domains of bacterial two-component regulatory systems in addition to the catalytic domain of adenylate cyclase (15,16). The possibility has been considered that the autophosphorylation of the transmitter domain and subsequent phosphotransfer to the receiver domains within the molecule regulates the catalytic activity.
In the present study, CyaC was shown to have the ability to autophosphorylate with [␥-32 P]ATP in vitro. On the other hand, the CyaC H572Q mutant was shown to be incapable of autophosphorylation. Thus, His 572 in the transmitter domain is thought to be the initial phosphorylation site in CyaC. Studies on the stability of phosphate links in phosphorylated CyaC derivatives showed that the wild-type and D60A mutant proteins contain both an acyl phosphate linkage and N-phosphate linkage, whereas the D895A mutant and the D60A,D895A double mutant CyaC contain an N-phosphate linkage alone. The fact that the acyl phosphate linkage in phosphorylated CyaC is lost when Asp 895 is replaced by an alanine residue indicates that Asp 895 is actually phosphorylated. Thus, after the autophosphorylation of His 572 in the transmitter domain, the phosphoryl group bound to His 572 is transferred to Asp 895 in the receiver (R2) domain. Asp 60 , the predicted phosphorylation site in the receiver (R1) domain, is not likely to be phosphorylated by the phosphotransfer reaction from the transmitter domain of CyaC. AsgA of Myxococcus xanthus, which is required for the formation of fruiting bodies, consists of a receiver domain on Aliquots (0.37 g) of purified wild-type or mutant His-CyaC proteins were phosphorylated, loaded onto an 8% SDS-PAGE gel for electrophoresis, and then transferred to an Immobilon-P membrane as described under "Experimental Procedures." The membrane was cut into three strips, and each strip was incubated in one of three solutions (50 mM Tris-HCl (pH 7.2) (black bars), 3 M NaOH (gray bars), or 1 M HCl (hatched bars)) for 1 h at 25°C. After treatment, all strips were washed briefly with distilled water, dried in air, and exposed to x-ray film. The remaining radioactivity was quantified by densitometry, and the results are presented as relative values. the N-terminal side and a transmitter domain on the C-terminal side. The primary structure of the two domains of AsgA is similar to the receiver (R1) domain and the transmitter domain of CyaC. It has been shown that AsgA autophosphorylates in the transmitter domain but that the phosphoryl group once bound to the transmitter domain is not transferred to the receiver domain. Plamann et al. (22) proposed that the AsgA receiver domain functions as an input domain for the transmitter. Interestingly, when a homology search was performed using the GenBank TM BLAST server, the receiver (R1) domain of CyaC showed especially high similarity to the receiver domain of AsgA in comparison with other receiver domains in response regulator proteins. The receiver (R1) domain of CyaC may have a function similar to that of the receiver domain of AsgA.
The adenylate cyclase activity of the CyaC H572Q mutant, which does not autophosphorylate, is about 4-fold lower than that of wild-type CyaC. Furthermore, the adenylate cyclase activities of the CyaC D895A mutant and CyaC D60A,D895A double mutant, which are able to autophosphorylate but unable to transfer the phosphoryl group from His 572 to Asp 895 , are at least 5-fold lower than that of wild-type CyaC. Thus the phos-phorylation of Asp 895 is required for the full activity of CyaC, consistent with the results that CyaC is phosphorylated at Asp 895 by acetyl phosphate (Fig. 6) and that the adenylate cyclase activity is stimulated in the presence of acetyl phosphate (Fig. 7). These results indicate that CyaC autophosphorylates first at His 572 in the transmitter domain, after which the phosphoryl group is transferred to Asp 895 in the receiver (R2) domain, and this stimulates the adenylate cyclase activity (Fig. 8).
Small phosphorylated molecules, such as acetyl phosphate, phosphoramidate, and carbamoyl phosphate, can act as phosphodonors for the phosphorylation of the receiver domains of response regulators in place of phosphorylated transmitter domains. Each response regulator protein shows different reactivities toward these small compounds (33). Spo0F, a response regulator protein in Bacillus subtilis, is not phosphorylated by acetyl phosphate in vitro while it is phosphorylated by a sensory kinase, KinA (35). In CyaC, the two receiver domains show different responses to acetyl phosphate; Asp 895 is phosphorylated by acetyl phosphate, while Asp 60 is not (Fig. 6). This probably reflects that the two aspartate residues have different physiological functions. It is probable that phosphoryl groups may be transferred to Asp 60 residue of the receiver (R1) domain from the transmitter domains of other sensory kinases that have not yet been identified. The autophosphorylation activity, the phosphotransfer from His 572 to Asp 895 , or the adenylate cyclase activity of CyaC may be regulated by the phosphorylation of Asp 60 .
We note that the phosphorylation levels observed in His-CyaC(wild-type) and His-CyaC(D60A), which are phosphorylated at both His 572 and Asp 895 , are lower than those observed in His-CyaC(D895A) and His-CyaC(D60A,D895A). In the receiver domain (R2) derivatives, phosphoryl groups accumulate at His 572 , because phosphoryl groups bound to His 572 are not transferred (Fig. 3). The low levels of phosphorylation observed in His-CyaC(wild-type) and His-CyaC(D60A) may be caused by dephosphorylation from phosphorylated Asp 895 . The phosphoryl groups of several phosphorylated response regulators are known to hydrolyze very rapidly. The half-life of hydrolysis of the phosphoaspartate group in phospho-CheY, a response regulator protein required for chemotaxis, is a few seconds (36). This instability is called autophosphatase activity. In addition, some sensory kinases are known to function to facilitate the rate of dephosphorylation of their cognate response regulators (37). CyaC also must have a mechanism to dephosphorylate its phosphorylated form. The rapid dephosphorylation from phosphorylated Asp 895 may be required to respond to subsequent signals. However, there is a possibility that the rate of autophosphorylation decreases when Asp 895 is phosphorylated.
It is noted that CyaC shows significant adenylate cyclase activity even if Asp 895 is not phosphorylated. This is similar to CheB and RegA, which are alternative proteins with the catalytic domain of an enzyme beside the receiver domain of the bacterial two-component regulatory systems (20,21). It is likely that the autophosphorylation activity of CyaC is regulated by sensing a specific signal that is transferred from a primary signal sensor of the cell. In sensory kinases, the Nterminal side of the transmitter domain is thought to be a signal input domain (18,19). The presumed signal input domain of CyaC, which was called the ETR1-like domain in the previous work (16), is similar to the ethylene sensor of Arabidopsis thaliana (ETR1) and to RcaE, the sensor for the complementary chromatic adaptation of Fremyella diplosiphon. This domain also exists in several other sensory kinases of Synechocystis PCC 6803 (17). Although the function of the ETR1-like domain remains unknown, it may participate in the recognition of a specific signal.
A CyaC homologue is found in the cyanobacterium Anabaena sp. strain PCC 7120 (15), in which cellular cAMP levels are reduced in response to a light-on signal. On the other hand, cAMP levels in the disruptant of the cyaC homologue are not affected by the light-on signal (15). It has been suggested that CyaC responds to a light signal; however, CyaC itself would not be a photoreceptor because it has no typical chromophore binding motifs in its amino acid sequence. Recently, a phytochrome that acts as a sensory photoreceptor in plants has been found in the cyanobacterium Synechocystis PCC 6803 (17,38). The cyanobacterial phytochrome consists of two functional domains, an N-terminal domain homologous to the chromophore attachment domain of the phytochrome and a C-terminal domain homologous to the transmitter domain of the sensory kinase. Such a photoreceptor having phosphorylation capability might control the activity of CyaC through the mechanism of a phosphotransfer reaction.