A defined subset of adenylyl cyclases is regulated by bicarbonate ion.

The molecular basis by which organisms detect and respond to fluctuations in inorganic carbon is not known. The cyaB1 gene of the cyanobacterium Anabaena sp. PCC7120 codes for a multidomain protein with a C-terminal class III adenylyl cyclase catalyst that was specifically stimulated by bicarbonate ion (EC50 9.6 mm). Bicarbonate lowered substrate affinity but increased reaction velocity. A point mutation in the active site (Lys-646) reduced activity by 95% and was refractory to bicarbonate activation. We propose that Lys-646 specifically coordinates bicarbonate in the active site in conjunction with an aspartate to threonine polymorphism (Thr-721) conserved in class III adenylyl cyclases from diverse eukaryotes and prokaryotes. Using recombinant proteins we demonstrated that adenylyl cyclases that contain the active site threonine (cyaB of Stigmatella aurantiaca and Rv1319c of Mycobacterium tuberculosis) are bicarbonate-responsive, whereas adenylyl cyclases with a corresponding aspartate (Rv1264 of Mycobacterium) are bicarbonate-insensitive. Large numbers of class III adenylyl cyclases may therefore be activated by bicarbonate. This represents a novel mechanism by which diverse organisms can detect bicarbonate ion.


Introduction
cAMP is one of the most prevalent signaling molecules among prokaryotes and eukaryotes, modulating the responses of an organism to diverse environmental stimuli.
The enzyme adenylyl cyclase (AC) 1 synthesizes cAMP and belongs to a large gene family consisting of six phylogenetically defined classes (1)(2)(3)(4). Class I ACs are found in the Enterobacteria e.g. Escherichia coli; class II ACs are exclusive to certain toxinproducing bacteria e.g. Bacillus anthracis; class III (the universal class) ACs are the only class found among higher eukaryotes and also includes the mammalian guanylyl cyclases and prokaryotic members; class IV enzymes are found in certain prokaryotic thermophiles e.g. Aeromonas hydrophila; class V consists of a single member from the obligate anaerobe Prevotella ruminicola; and the recently described class VI ACs found in the genomes of the Rhizobiaceae.
cAMP is synthesized in mammals by a seemingly ubiquitous family of class III plasma membrane spanning ACs (transmembrane adenylyl cyclase; tmAC), which mediates cellular responses to extracellular signals. Additionally, a cytosolic form of AC (soluble adenylyl cyclase; sAC) has been identified in mammals that was demonstrated to be molecularly and biochemically distinct from the tmACs (5). Although most abundantly expressed in testis, sAC is expressed ubiquitously (6,7) and is directly activated by bicarbonate ion in a pH independent manner (8).
The HCO 3 regulated mammalian sAC is more closely related to other prokaryotic class III ACs than to other mammalian tmACs (5,9) Consistent with this phylogenetic relationship, it was demonstrated that a single cyanobacterial class III AC, cyaC of Spirulina platensis, was also stimulated by HCO 3 - (8). If HCO 3 stimulation were a general feature of at least a subset of class III ACs they would represent the first family 4 of HCO 3 responsive signaling molecules. HCO 3 is fundamental to prokaryotic biology; accumulated cytoplasmic HCO 3 is the primary source of inorganic carbon transported to the cyanobacterial carboxysome for photosynthesis (10) and is also hypothesized to have been the predominant carbon source utilized by oxygenic phototrophs in the generation of Earth's oxygen atmosphere (11).
To define the extent to which class III ACs may be stimulated by HCO 3 -

Recombinant DNAs
The cyaB1 gene of Anabaena sp. PCC7120 with associated single amino acid point mutations and the Mycobacterium tuberculosis H37Rv Rv1264 gene were assembled as previously described (14,15). Full details of the Mycobacterium Rv1319c gene will be reported elsewhere. Nucleotides

Expression and purification of bacterially expressed proteins
Anabaena cyaB1 wild type and mutant proteins and Mycobacterium Rv1264 1-397 protein were expressed and purified as previously described (14,15). Full details of the Mycobacterium Rv1319c protein will be reported elsewhere.
The Stigmatella pQE30-c y a B construct was transformed into E .

AC assay
The AC activity of cyaB1 wild type protein, cyaB1 mutant proteins, and other prokaryotic  the ACs ( Figure 1A). T721, a residue essential for full catalysis in cyaB1 (14) was conserved among several of the ACs including HCO 3 responsive sAC and Spirulina cyaC, while the remainder expressed a D residue essential for substrate definition in the corresponding position. Given the conservation of the active site T polymorphism between cyaB1, sAC, and Spirulina cyaC we investigated whether cyaB1 was also stimulated by HCO 3 -. We expressed the catalytic domain of cyaB1 (cyaB1 595-859 ) to include a region of the C-terminus (amino acids 795-828) that had some similarity to a tetratricopeptide repeat and is essential for production of functional soluble protein in Escherichia coli (14).
The activity of cyaB1 595-859 was measured in the presence or absence of various salts ( Figure 1B). Specific activity was unchanged in the presence of NaCl and KCl while NaHCO 3 and KHCO 3 both gave an approximately two-fold increase of cyaB1 595-859 specific activity demonstrating that HCO 3 activation of cyaB1 595-859 was independent of the associated cation. We measured the specific activity of cyaB1 595-859 over a range of HCO 3 concentrations with Clas a control for non-specific ionic effects ( Figure 2A). A maximal two-fold stimulation was seen in the presence of HCO 3 with an EC 50 of 9.6 mM.
The GAF-B domain of cyaB1 binds cAMP and activates the AC catalytic domain (14). 8 cyaB1 therefore acts as a self-activating switch. We asked whether the behavior of this switch is affected by HCO 3 and expressed recombinant protein corresponding to the cyaB1 holoenzyme (cyaB1  ) that contains the GAF domains and examined its specific activity in the presence or absence of HCO 3 -. cyaB1 1-859 specific activity showed a nonlinear time dependence as previously reported (14) and the rate of cAMP formation was significantly accelerated in the presence of 10 mM KHCO 3 indicating that HCO 3 activated the GAF-B mediated positive feedback mechanism of cyaB1 ( Figure 2B). The rate of cAMP formation was also stimulated in the presence of 10 mM NaHCO 3 , but inhibited in the presence of higher concentrations of NaHCO 3 indicating that Na + may block GAF-B binding of cAMP or intramolecular signalling 2 .
cyaB1 595-859 specific activity showed a non-linear protein dependence ( Figure 3) indicating that homodimerization was necessary for formation of the active site. This has been independently confirmed by titration of complementary mutant cyaB1 595-859 proteins that are inactive as homodimers, but restored catalytic activity as heterodimers (14). To determine whether HCO 3 up regulated cyaB1 595-859 specific activity by increasing homodimer formation we examined the ratio of the HCO 3 and Clspecific activities as a function of protein concentration. Interestingly, this ratio remained constant over the range of protein concentrations tested indicating that HCO 3 did not affect homodimer formation. The protein concentration independence of HCO 3 up regulation of specific activity allowed us to make comparisons between experiments in which different concentrations of protein were assayed (see Figure 4).  [17]) with a threonine (T721 in cyaB1). D1018 is involved in substrate definition in AC by forming a hydrogen bond with N 6 of the adenine ring of ATP (17). T721 functionally replaced this aspartate and may act as a hydrogen acceptor from the purine ring (14). When assayed at pH 7.5 to eliminate problems with divalent metal ion depletion, cyaB1 595-859 specific activity was stimulated approximately three-fold relative to the Clactivity over the tested range (0-60 mM HCO 3 -) ( Figure 4A). We investigated the involvement of the canonical active site residues of a class III AC in HCO 3 stimulation using point mutations. Although the basal specific activities of cyaB1 595-859 R732A (transition state stabilization), cyaB1 595-859 N728A (transition state stabilization), and cyaB1 595-859 D719A (a residue examined for possible functional homology to D1018 of AC IIC 2 ) were significantly reduced compared to wild type enzyme their fold stimulation by HCO 3 was equivalent (Supplemental Data Figure 1). A key difference between T721 of cyaB1 and D1018 of AC IIC 2 is the loss of the aspartate carboxy group. We reasoned that HCO 3 possibly mimics the carboxy group within the active site but, interestingly, 10 HCO 3 mediated up regulation of cyaB1 595-859 T721A specific activity was equivalent to wild type despite a >99% reduction in basal activity ( Figure 4B). We noted that K938 of AC IIC 2 (substrate definition and equivalent to K646 of cyaB1; [17]) was proposed to act not only as a hydrogen acceptor for the N 1 of the ATP purine ring but also as a hydrogen donor to the carboxy group of the adjacent D1018 residue (19). Thus K646 may form a stabilizing hydrogen bond with HCO 3 at a position equivalent to the carboxy group of AC IIC 2 . Although basal activity was reduced by approximately 95%, HCO 3 activation was completely abolished in cyaB1 595-859 K646A in support of this hypothesis ( Figure 4C). If Although the amino acid equivalent to K646 of cyaB1 and K938 of AC IIC 2 is conserved in all the ACs examined ( Figure 1A) we reasoned that an adjacent threonine or aspartate within the catalytic cleft of a class III enzyme (i.e. at the position corresponding to T721) could be a marker for HCO 3 -AC responsiveness or nonresponsiveness, respectively. To test this hypothesis we generated recombinant proteins corresponding to diverse prokaryotic class III ACs with either a threonine or aspartate at the position equivalent to cyaB1 T721 ( Figure 1A) and examined them for their response to HCO 3 -.
Stigmatella aurantiaca B17R20 is a myxobacterium from which two ACs have been identified (20). We expressed amino acids 160 to 353 of cyaB as a recombinant protein (cyaB 160-353 ) that contained a threonine residue (T293) at the position corresponding to cyaB1 T721 ( Figure 1A). cyaB 160-353 specific activity was up regulated by HCO 3 approximately two-fold relative to the Cldependent activity (EC 50 8.6 mM) 11 ( Figure 5A) consistent with the hypothesis that the threonine at amino acid 293 is a marker for HCO 3 responsiveness. This stimulation was maintained in the presence of alternative anions to Clindicating that cyaB 160-353 was most likely stimulated by HCO 3 rather than inhibited by Cl -2 .
Mycobacterium tuberculosis H37Rv is a gram-negative bacterium and important human pathogen for which the genome has revealed a number of putative class III ACs (15,21,22). We expressed two ACs that contain either a threonine (amino acids 356-535 of Rv1319c) or an aspartate (Rv1264 holoenzyme) at the position corresponding to T721 of cyaB1 ( Figure 1A). Consistent with our hypothesis that the threonine residue is a marker for AC HCO 3 responsiveness Rv1319c 356-535 specific activity was up regulated approximately three-fold in the presence of HCO 3 over the concentration range tested ( Figure 5B) while Rv1264 1-397 specific activity did not respond to HCO 3 over an identical concentration range ( Figure 5C).
The data of Figure 5 supports the hypothesis posited in Figure 4 and indicates that HCO 3 responsive class III AC domains are widespread in biology and represents the sole candidate mechanism for HCO 3 detection in an organism.

Discussion
cyaB1 of Anabaena sp. PCC7120 is a class III AC whose catalytic center is functionally equivalent to that identified for the mammalian tmACs (17,18) except for a threonine residue (T721) which replaces an aspartate highly conserved among the tmACs. T721 functionally replaces aspartate and is suggested to act as a hydrogen acceptor from the purine ring (14). cyaB1 catalytic activity was demonstrated to be responsive to HCO 3 extending the number of identified class III ACs that are stimulated by HCO 3 and stimulation was cation independent and anion dependent. The measured EC 50 of 9.6 mM is well within the range of calculated intracellular HCO 3 concentrations for cyanobacteria (23). Although the inorganic carbon pool for Anabaena sp. PCC7120 has not been measured, the related heterocyst forming species Anabaena variabilis M3 can accumulate up to 50 mM internal inorganic carbon depending upon the growth conditions (24). cAMP production through cyaB1 is therefore likely to be responsive to variations in intracellular HCO 3 -. Intracellular cAMP has previously been correlated with the rate of HCO 3 uptake in the cyanobacterium Anabaena flos-aquae (25) indicating that the protein chemistry we describe is functional in vivo. HCO 3 was able to functionally activate not only the catalytic domains but also the entire holoenzyme with its associated GAF and PAS domains. The GAF-B mediated positive feedback loop created by cyaB1 may therefore be accelerated by the availability of a fixable carbon source in Anabaena sp. PCC7120.
HCO 3 did not affect cyaB1 homodimer formation or lower the activation energy for transition state formation but did significantly alter substrate binding kinetics by increasing the K M for ATP and V max . The cyanobacterium Synechococcus PCC6301 (Anacystis nidulans) has an intracellular ATP concentration of approximately 1 mM (value calculated from data in [26]). As the K M (ATP) for both cyaB1 595-859 and holoenzyme is of the order of <50 µM it is likely that the effect of HCO 3 on K M is biologically irrelevant 13 and that cyaB1 is activated by HCO 3 in the intracellular environment by an increase in reaction velocity. Point mutations revealed that loss of T721 did not affect cyaB1 595-859 HCO 3 responsiveness. We demonstrated, however, that loss of K646 (equivalent to K938 of AC IIC 2 ) ablated HCO 3 stimulation of specific activity. In class III ACs that contain an aspartate residue corresponding to the position of T721, the adjacent lysine in the catalytic center has been proposed to form a hydrogen bond with the aspartate carboxy group (19). We hypothesize that in cyaB1 HCO 3 can functionally replace this carboxy group and is co-ordinated within the catalytic cleft by K646. A T721D point mutation was refractory to HCO 3 in support of this hypothesis. The enhanced basal activity of T721D relative to T721A may represent an enzyme mimicking HCO 3 activation. If HCO 3 does functionally replace the carboxy group of an aspartate, it is surprising that HCO 3 increases K M (ATP) given that a logical extension of our hypothesis would be that HCO 3 forms a hydrogen bond with N 6 of the adenine ring and increase affinity for substrate. It is possible that HCO 3 binding results in subtle changes in the structure of the substrate-binding pocket that lowers affinity, but optimizes orientation for catalysis. As there is no effect on E a in the presence of HCO 3 it is unlikely that this effect is on the acquisition of the transition state. The increase in k cat demonstrates that there is an increase in catalytic activity on formation of the enzyme-substrate complex and this may therefore occur after formation of the transition state. The exact mechanism of HCO 3 activation of AC is an interesting question that requires further investigation.