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• W2142094277 abstract "Carbon dioxide is fundamental to the physiology of all organisms. There is considerable interest in the precise molecular mechanisms that organisms use to directly sense CO2. Here we demonstrate that a mammalian recombinant G-protein-activated adenylyl cyclase and the related Rv1625c adenylyl cyclase of Mycobacterium tuberculosis are specifically stimulated by CO2. Stimulation occurred at physiological concentrations of CO2 through increased kcat. CO2 increased the affinity of enzyme for metal co-factor, but contact with metal was not necessary as CO2 interacted directly with apoenzyme. CO2 stimulated the activity of both G-protein-regulated adenylyl cyclases and Rv1625c in vivo. Activation of G-protein regulated adenylyl cyclases by CO2 gave a corresponding increase in cAMP-response element-binding protein (CREB) phosphorylation. Comparison of the responses of the G-protein regulated adenylyl cyclases and the molecularly, and biochemically distinct mammalian soluble adenylyl cyclase revealed that whereas G-protein-regulated enzymes are responsive to CO2, the soluble adenylyl cyclase is responsive to both CO2 and bicarbonate ion. We have, thus, identified a signaling enzyme by which eukaryotes can directly detect and respond to fluctuating CO2. Carbon dioxide is fundamental to the physiology of all organisms. There is considerable interest in the precise molecular mechanisms that organisms use to directly sense CO2. Here we demonstrate that a mammalian recombinant G-protein-activated adenylyl cyclase and the related Rv1625c adenylyl cyclase of Mycobacterium tuberculosis are specifically stimulated by CO2. Stimulation occurred at physiological concentrations of CO2 through increased kcat. CO2 increased the affinity of enzyme for metal co-factor, but contact with metal was not necessary as CO2 interacted directly with apoenzyme. CO2 stimulated the activity of both G-protein-regulated adenylyl cyclases and Rv1625c in vivo. Activation of G-protein regulated adenylyl cyclases by CO2 gave a corresponding increase in cAMP-response element-binding protein (CREB) phosphorylation. Comparison of the responses of the G-protein regulated adenylyl cyclases and the molecularly, and biochemically distinct mammalian soluble adenylyl cyclase revealed that whereas G-protein-regulated enzymes are responsive to CO2, the soluble adenylyl cyclase is responsive to both CO2 and bicarbonate ion. We have, thus, identified a signaling enzyme by which eukaryotes can directly detect and respond to fluctuating CO2. Inorganic carbon (Ci) 3The abbreviations used are: Ci, inorganic carbon; AC, adenylyl cyclase; sAC, soluble AC; CREB, cAMP-response element-binding protein; Mes, 2-[N-morpholino]ethanesulfonic acid; Mops, 3-[N-morpholino]propanesulfonic acid; GTPγS, guanosine 5′-3-O-(thio)triphosphate. is central to prokaryotic and eukaryotic physiology. The predominant biologically active forms of Ci are CO2 and HCO3- and their relative contributions to the total Ci pool are pH-dependent. Biological roles for CO2 and HCO3- include photosynthetic carbon fixation (1Falkowski P.G. Raven J.A. Aquatic Photosynthesis. 2nd Ed. Princeton University Press, Princeton2007: 156-200Google Scholar), pH homeostasis (2Roos A. Boron W.F. Physiol. Rev. 1981; 61: 296-434Crossref PubMed Scopus (2297) Google Scholar), carbon metabolism (3Smith K.S. Ferry J.G. FEMS Microbiol. Rev,. 2000; 24: 335-366Crossref PubMed Google Scholar), activation of virulence in pathogenic organisms (4Bahn Y.S. Muhlschlegel F.A. Curr. Opin. Microbiol. 2006; 9: 572-578Crossref PubMed Scopus (112) Google Scholar), sperm maturation (5Esposito G. Jaiswal B.S. Xie F. Krajnc-Franken M.A. Robben T.J. Strik A.M. Kuil C. Philipsen R.L. van Duin M. Conti M. Gossen J.A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2993-2998Crossref PubMed Scopus (321) Google Scholar), and as an alarmone in Drosophila (6Jones W.D. Cayirlioglu P. Kadow I.G. Vosshall L.B. Nature. 2007; 445: 86-90Crossref PubMed Scopus (481) Google Scholar, 7Kwon J.Y. Dahanukar A. Weiss L.A. Carlson J.R. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 3574-3578Crossref PubMed Scopus (357) Google Scholar). Given its importance in biology, the identification of CO2 responsive signaling pathways is key to understanding how organisms cope with fluctuating CO2. Two seven transmembrane receptors, Gr21a and Gr63a, have been shown to confer CO2 responsiveness in Drosophila neurons (6Jones W.D. Cayirlioglu P. Kadow I.G. Vosshall L.B. Nature. 2007; 445: 86-90Crossref PubMed Scopus (481) Google Scholar, 7Kwon J.Y. Dahanukar A. Weiss L.A. Carlson J.R. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 3574-3578Crossref PubMed Scopus (357) Google Scholar). Guanylyl cyclase D expressing olfactory neurons also mediate sensitivity to CO2 in mice (8Hu J. Zhong C. Ding C. Chi Q.Y. Walz A. Mombaerts P. Matsunami H. Luo M.M. Science. 2007; 317: 953-957Crossref PubMed Scopus (190) Google Scholar). A role for cGMP-activated channels in CO2 sensing has been observed in CO2 avoidance behavior in Caenorhabditis (9Bretscher A.J. Busch K.E. de Bono M. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 8044-8049Crossref PubMed Scopus (122) Google Scholar, 10Hallem E.A. Sternberg P.W. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 8038-8043Crossref PubMed Scopus (145) Google Scholar). Despite these impressive advances, no eukaryotic signaling enzymes unequivocally demonstrated to respond to CO2 have been identified. The mammalian soluble adenylyl cyclase (sAC) synthesizes the second messenger 3′,5′-cAMP and is directly stimulated by HCO3- (11Buck J. Sinclair M.L. Schapal L. Cann M.J. Levin L.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 79-84Crossref PubMed Scopus (426) Google Scholar, 12Chen Y. Cann M.J. Litvin T.N. Iourgenko V. Sinclair M.L. Levin L.R. Buck J. Science. 2000; 289: 625-628Crossref PubMed Scopus (690) Google Scholar, 13Jaiswal B.S. Conti M. J. Biol. Chem. 2001; 276: 31698-31708Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Stimulation of sAC by HCO3- has an unequivocal role in sperm maturation (5Esposito G. Jaiswal B.S. Xie F. Krajnc-Franken M.A. Robben T.J. Strik A.M. Kuil C. Philipsen R.L. van Duin M. Conti M. Gossen J.A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2993-2998Crossref PubMed Scopus (321) Google Scholar, 14Hess K.C. Jones B.H. Marquez B. Chen Y. Ord T.S. Kamenetsky M. Miyamoto C. Zippin J.H. Kopf G.S. Suarez S.S. Levin L.R. Williams C.J. Buck J. Moss S.B. Dev. Cell. 2005; 9: 249-259Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 15Xie F. Conti M. Dev. Biol. 2004; 265: 196-206Crossref PubMed Scopus (30) Google Scholar, 16Xie F. Garcia M.A. Carlson A.E. Schuh S.M. Babcock D.F. Jaiswal B.S. Gossen J.A. Esposito G. van Duin M. Conti M. Dev. Biol. 2006; 296: 353-362Crossref PubMed Scopus (191) Google Scholar). sAC is a member of the Class III family of adenylyl cyclases (ACs), a family that also includes the G-protein-regulated ACs and many examples from prokaryotic genomes (17Baker D.A. Kelly J.M. Mol. Microbiol. 2004; 52: 1229-1242Crossref PubMed Scopus (105) Google Scholar, 18Sunahara R.K. Taussig R. Mol. Interv. 2002; 2: 168-184Crossref PubMed Scopus (340) Google Scholar). The Class III ACs can be divided into four subclasses (a–d) based upon polymorphisms within the active site (19Linder J.U. Schultz J.E. Cell. Signal. 2003; 15: 1081-1089Crossref PubMed Scopus (145) Google Scholar). sAC is a member of Class IIIb, a subclass characterized partly by replacement of a substrate binding Asp with Thr. The Class IIIa ACs include the mammalian G-protein-stimulated ACs and numerous prokaryotic examples. These have been previously assumed to be non-responsive to Ci (12Chen Y. Cann M.J. Litvin T.N. Iourgenko V. Sinclair M.L. Levin L.R. Buck J. Science. 2000; 289: 625-628Crossref PubMed Scopus (690) Google Scholar). All prokaryotic Class IIIb ACs examined to date respond to Ci including enzymes from organisms as diverse as Anabaena PCC 7120, Mycobacterium tuberculosis, Stigmatella aurantiaca, and Chloroflexus aurantiacus (20Cann M.J. Hammer A. Zhou J. Kanacher T. J. Biol. Chem. 2003; 278: 35033-35038Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 21Kobayashi M. Buck J. Levin L.R. Dev. Genes Evol. 2004; 214: 503-509PubMed Google Scholar). Two Class IIIb ACs, Slr1991 of Synechocystis PCC 6803 and CyaB1 of Anabaena PCC 7120, have been proven to respond to CO2 and not HCO3-, giving rise to the idea of AC as a true gas-sensing molecule (22Hammer A. Hodgson D.R. Cann M.J. Biochem. J. 2006; 396: 215-218Crossref PubMed Scopus (41) Google Scholar, 23Raven J.A. Biochem. J. 2006; 396: e5-7Crossref PubMed Scopus (13) Google Scholar). The finding that Class IIIb ACs respond to CO2 and not HCO3- necessitates an examination of the assumption that G-protein-regulated ACs and related prokaryotic enzymes do not respond to Ci. Here we demonstrate, contrary to previous work, that a recombinant G-protein-regulated AC and the Class IIIa Rv1625c AC of M. tuberculosis H37Rv show a pH-dependent response to Ci due to specific stimulation by CO2 at physiologically relevant concentrations. CO2 interacted directly with the apoprotein and modulated the activity of both the prokaryotic enzyme and G-protein-regulated AC in vivo. Finally, we contrasted the responses of sAC- and G-protein-regulated ACs to different species of Ci and propose that the mammalian cAMP signaling pathway is able to discriminate between CO2 and HCO3- in vivo. Recombinant Proteins—Rv1625c204–443 wild type and mutant proteins, Slr1991120–337 wild type and mutant proteins, recombinant protein corresponding to amino acids 1–469 of human sAC (truncated splice variant (13Jaiswal B.S. Conti M. J. Biol. Chem. 2001; 276: 31698-31708Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar); sACT), recombinant protein corresponding to the first catalytic domain (amino acids 263–476; 7C1) of human AC type 7, and recombinant protein corresponding to the second catalytic domain (amino acids 821–1090; 2C2) of rat AC type 2 were expressed and purified as previously described (22Hammer A. Hodgson D.R. Cann M.J. Biochem. J. 2006; 396: 215-218Crossref PubMed Scopus (41) Google Scholar, 24Dessauer C.W. Gilman A.G. J. Biol. Chem. 1996; 271: 16967-16974Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 25Guo Y.L. Seebacher T. Kurz U. Linder J.U. Schultz J.E. EMBO J. 2001; 20: 3667-3675Crossref PubMed Scopus (91) Google Scholar, 26Litvin T.N. Kamenetsky M. Zarifyan A. Buck J. Levin L.R. J. Biol. Chem. 2003; 278: 15922-15926Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 27Yan S.Z. Tang W.J. Methods Enzymol. 2002; 345: 231-241Crossref PubMed Scopus (8) Google Scholar). A mixture of 7C1 with an excess of 2C2 (7C1·2C2) represents a catalytically active G-protein responsive AC without the transmembrane domains of the native molecule. Recombinant protein representing the short splice variant of bovine Gsα was purified and activated with GTPγS·Mg2+ as previously described (28Lee E. Linder M.E. Gilman A.G. Methods Enzymol. 1994; 237: 146-164Crossref PubMed Scopus (247) Google Scholar). Single amino acid mutations were introduced by site-directed mutagenesis using appropriate primers and the appropriate wild type construct as template. Double amino acid mutations were introduced by site-directed mutagenesis using appropriate primers and the appropriate single amino acid mutant construct as template. All constructs were confirmed by double-stranded sequencing. Mutagenic primer sequences are provided in Table S1. Plasmids encoding Rv1625c204–443 K296A and D256A mutagenic proteins were a kind gift of Joachim Schultz (25Guo Y.L. Seebacher T. Kurz U. Linder J.U. Schultz J.E. EMBO J. 2001; 20: 3667-3675Crossref PubMed Scopus (91) Google Scholar). Adenylyl Cyclase Assays—AC assays were performed at 37 °C (Rv1625c204–443) or30 °C (7C1·2C2) in a final volume of 100 μl and contained 50 mm buffer, 2 mm [2,8-3H]cAMP (150 Bq), and [α-32P]ATP (25 kBq) if not stated otherwise (29Salomon Y. Londos C. Rodbell M. Anal. Biochem. 1974; 58: 541-548Crossref PubMed Scopus (3374) Google Scholar). Protein concentrations were adjusted to maintain substrate utilization at <10%. The following buffers were used at pH 6.5 (Mes), pH 7.0–7.5 (Mops), and pH 8.0–8.5 (Tris-hydrochloride). Enzyme, buffer, and substrate were prepared at the appropriate pH. CO2 was quantified by titration against NaOH. Assay pH was stable over a period of at least 40 min. For dose-response experiments, NaHCO3 was added to the assay, and the CO2 concentration was calculated using the Henderson-Hasselbalch equation, and the total salt concentration was adjusted with NaCl. All errors correspond to the S.E. If absent, errors were smaller than the symbol used to depict the data point. Adenylyl Cyclase Assays at Ci Disequilibrium—For Ci disequilibrium assays, dissolved CO2 was prepared by bubbling into double-distilled H2O at 0 °C to saturation and quantified by titration against NaOH. NaHCO3 and NaCl were prepared in double-distilled H2O at 0 °C. CO2, HCO3-, or Cl– were subsequently added to the assay at 0 °C simultaneous with substrate to the required concentration. Buffer and substrate for assays were prepared at the appropriate pH and temperature for the experiment. pH changes in assays were monitored using a pH electrode (Biotrode; Hamilton) connected to a computer with a PC card (Orion Sensorlink). The pH was measured in a time-driven acquisition mode in assays identical to those used for biochemistry. All pH measurements were accurate to ± 0.02 pH units (manufacturers specifications). All errors correspond to the S.E. CO2 Activation of AC in Vivo—pCTXLacZ, a plasmid with lacZ expression driven from a cAMP-responsive promoter, and pQE30-Rv1625c204–443 (25Guo Y.L. Seebacher T. Kurz U. Linder J.U. Schultz J.E. EMBO J. 2001; 20: 3667-3675Crossref PubMed Scopus (91) Google Scholar) were transformed into Escherichia coli M15 (pREP4). Cells were grown in Luria broth with 100 μg ml–1 ampicillin, 50 μgml–1 kanamycin, and 5 μgml–1 tetracycline at 30 °C until an A600 of 0.6. Rv1625c204–443 protein production was induced with 30 μm isopropyl 1-thio-β-d-galactopyranoside for 3 h. Cells were pelleted at 4000 × g for 10 min and resuspended in Luria broth containing 50 mm Tris, pH 7.1. Cell suspensions were bubbled with either 10% (v/v) CO2 in air or in air for 30 min at 30 °C. Cells were disrupted with 0.1 mg of sodium deoxycholate and 1% (v/v) toluene and mixed for 10 min at 30 °C. The lysate was made up to 50 mm sodium phosphate, pH 7.0, 0.5 mm ortho-nitrophenol-β-d-galactopyranoside and incubated for 15 min at 30 °C. Reactions were stopped with 2 m sodium carbonate, and absorbance was read at 420 nm. A standard curve was generated using 0–250 μm ortho-nitrophenol. CO2 Binding Assays—1 ml of 50% (v/v) Sephadex G50 in 50 mm Mes, pH 6.5 (bed volume 0.5 ml), was pre-spun at 1500 × g for 30 s. A freshly prepared binding reaction of 23 nmol of protein, 30 mm NaH14CO3, pH 6.5, and 50 mm Mes, pH 6.5, (total volume 50 μl) was immediately added and centrifuged at 1500 × g for 30 s, and the flow-through collected into 50 μl of 2 m NaOH. Scintillation counting was used to measure 14C counts in the flow-through. Measurement of Intracellular pH—HEK 293T cells attached to a 24-mm diameter glass coverslip were loaded with the pH-sensitive fluorescent dye 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) through exposure to 1 μm BCECF-AM (an acetoxymethyl ester derivative) for 30 min. Intracellular pH was measured by exciting a small patch of cells at 490 and 440 nm using a microspectroflourometric system and measuring emission at 535 nm. pHi was calibrated using the high potassium nigericin method (30Hegyi P. Rakonczay Z. Gray M.A. Argent B.E. Pancreas. 2004; 28: 427-434Crossref PubMed Scopus (42) Google Scholar). cAMP Accumulation in Vivo—HEK 293T cells were cultured in 12-well plates and labeled overnight with 1.5 μCi of [3H]adenine at 80–90% confluence. Cells were washed with phosphate-buffered saline solution and incubated at the required CO2 concentration in preincubation media (10 mm HEPES-NaOH, 117 mm NaCl, 4.5 mm KCl, 1 mm MgCl2, 11 mm glucose, 10 mm sucrose, and 2.5 mm CaCl2) containing 1 mm isobutylmethylxanthine. Preincubation mixes were pre-gassed with the desired CO2 concentration and adjusted to pH 7.0. The assay was initiated after 30 min by the addition of agonist and incubated at the required CO2 concentration. Assays were stopped with 5% (w/v) trichloroacetic acid containing 1 mm ATP and 1 mm cAMP. Products were quantified by twin column chromatography (29Salomon Y. Londos C. Rodbell M. Anal. Biochem. 1974; 58: 541-548Crossref PubMed Scopus (3374) Google Scholar). For immunoblotting, samples were harvested after treatment as above except in the absence of [3H]adenine and isobutylmethylxanthine. Immunoblotting was performed using standard methodologies with anti-phospho-CREB (Ser133) and anti-α-tubulin as load control. The M. tuberculosis H37Rv genome contains at least 15 putative ACs and one cAMP phosphodiesterase, suggesting an important role for cAMP in the physiology of Mycobacterium (31Shenoy A.R. Capuder M. Draskovic P. Lamba D. Visweswariah S.S. Podobnik M. J. Mol. Biol. 2007; 365: 211-225Crossref PubMed Scopus (55) Google Scholar, 32Shenoy A.R. Sreenath N. Podobnik M. Kovacevic M. Visweswariah S.S. Biochemistry. 2005; 44: 15695-15704Crossref PubMed Scopus (60) Google Scholar, 33Shenoy A.R. Visweswariah S.S. Trends Microbiol. 2006; 14: 543-550Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 34Shenoy A.R. Visweswariah S.S. FEBS Lett. 2006; 580: 3344-3352Crossref PubMed Scopus (45) Google Scholar). cAMP is implicated in the pathogenesis of mycobacteria, and CO2 has been suggested as a signal to enable Mycobacterium to avoid phagosomal acidification (35Lowrie D.B. Aber V.R. Jackett P.S. J. Gen Microbiol. 1979; 110: 431-441Crossref PubMed Scopus (59) Google Scholar, 36Lowrie D.B. Jackett P.S. Ratcliffe N.A. Nature. 1975; 254: 600-602Crossref PubMed Scopus (80) Google Scholar). The Rv1625c gene of M. tuberculosis encodes an enzyme consisting of six putative transmembrane helices and a single Class IIIa AC catalytic domain (25Guo Y.L. Seebacher T. Kurz U. Linder J.U. Schultz J.E. EMBO J. 2001; 20: 3667-3675Crossref PubMed Scopus (91) Google Scholar, 37Reddy S.K. Kamireddi M. Dhanireddy K. Young L. Davis A. Reddy P.T. J. Biol. Chem. 2001; 276: 35141-35149Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The predicted topology, therefore, resembles one-half of a mammalian G-protein-regulated AC enzyme. A further similarity arises in the active site where six key catalytic residues distributed among the two catalytic domains of the G-protein-regulated ACs are present in Rv1625c to generate a homodimeric enzyme with two active sites (Fig. 1a). The Class IIIa Rv1625c AC was reported to be insensitive to Ci under experimental conditions where HCO3- was the predominant form of Ci. We expressed the AC domain of Rv1625c as a recombinant protein (Rv1625c204–443) and investigated the response of enzyme to constant Ci at varying pH (Fig. 1b). Relative stimulation (Ci:Cl–) varied from less than 1 at pH 8.5 (0.1 mm CO2, 19.6 mm HCO3-, 0.3 mm CO2–3) to 6.3 at pH 6.5 (7.7 mm CO2, 12.3 mm HCO3-). Stimulation of Rv1625c specific activity was most evident below pH 7.5, explaining a failure to previously observe a stimulation with Ci (20Cann M.J. Hammer A. Zhou J. Kanacher T. J. Biol. Chem. 2003; 278: 35033-35038Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). A requirement for low pH to observe a response to Ci is consistent with a role for CO2 as the activating species but may also be due to the altered protonation status of Rv1625c204–443 limiting the ability of the enzyme to respond to HCO3- at higher pH. We, therefore, assayed Rv1625c204–443 under conditions of Ci disequilibrium to determine whether CO2 or HCO3- is the activating species (22Hammer A. Hodgson D.R. Cann M.J. Biochem. J. 2006; 396: 215-218Crossref PubMed Scopus (41) Google Scholar, 38Cooper T.G. Filmer D. J. Biol. Chem. 1969; 244: 1081-1083Abstract Full Text PDF PubMed Google Scholar). AC assays performed under conditions of Ci disequilibrium exploit the fact that acquisition of the CO2/HCO3- equilibrium is significantly slowed at low temperature. We defined conditions for assaying AC under conditions of disequilibrium by following the acquisition of the CO2/HCO3- equilibrium through measuring the pH of a weakly buffered (5 mm) Mes solution on the addition of 20 mm CO2 or NaHCO3 in the presence or absence of carbonic anhydrase at 0 °C (data not shown). 4P. D. Townsend, P. M. Holliday, D. R. W. Hodgson, and M. J. Cann, unpublished observations. In this manner we defined conditions for assaying AC under conditions of disequilibrium using 20 mm CO2 or HCO3- as a 10-s assay period at 0 °C after the addition of Ci. Under these conditions, Ci is predominantly in the form added to the assay (CO2 or HCO3-) and has not significantly advanced toward the equilibrium determined by assay pH (clamped with 100 mm Mes in the actual AC assays). Control experiments demonstrated that under the conditions used for the assay final pH was equivalent when either CO2, HCO3-, or Cl– were added, demonstrating that any observed stimulation was due to addition of Ci and not a change in assay pH (Fig. 1c; inset). Ci disequilibrium assays proved that Rv1625c204–443 responded to CO2 and not HCO3- (Fig. 1c). This demonstrates that a Class IIIa AC is able to respond to Ci and confirms that the response is to CO2, as with Class IIIb ACs. Given the similarity in response to CO2 seen in Rv1625c204–443 and Class IIIb ACs, we examined the kinetic parameters for Rv1625c and compared them to the Class IIIb ACs (Table 1). CO2 stimulated Rv1625c204–443 specific activity through an increase in kcat, similar to findings with Class IIIb ACs, supporting the idea that the two subclasses share a similar mechanism of response to CO2 (20Cann M.J. Hammer A. Zhou J. Kanacher T. J. Biol. Chem. 2003; 278: 35033-35038Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 22Hammer A. Hodgson D.R. Cann M.J. Biochem. J. 2006; 396: 215-218Crossref PubMed Scopus (41) Google Scholar). A dose-response curve with increasing Ci revealed a 5-fold stimulation at 11.6 mm CO2 (Fig. 1d). Concentrations over 12 mm caused a gradual decrease in specific activity from this peak, making an EC50 impossible to unambiguously calculate. Stimulation was significant to a 95% confidence interval at 1.9 mm CO2.TABLE 1Kinetic parameters for Rv1625c204–443 and 7C1·2C2ParameterRv1625204-4437C1·2C2Cl-CO2Cl-CO2Vmax (nmol of cAMP mg-1 min-1)30.4 ± 0.876.0 ± 2.844.9 ± 2.876.4 ± 4.1Km[ATP] (mm) (S.D.)0.54 ± 0.021.72 ± 0.091.89 ± 0.252.04 ± 0.23kcat (s-1)5.914.66.410.7 Open table in a new tab Given the clear relationship between Rv1625c and the Class IIIb ACs with respect to the kinetics of activation in response to CO2, we investigated the activation mechanism. Mutation of a key substrate determining lysine (Lys-646) in the Class IIIb CyaB1 AC of Anabaena ablated the response of the enzyme to CO2 (20Cann M.J. Hammer A. Zhou J. Kanacher T. J. Biol. Chem. 2003; 278: 35033-35038Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). We generated recombinant protein for the corresponding mutation in Rv1625c (K296A) and assessed its response to CO2. Surprisingly, Rv1625c204–443 K296A retained responsiveness to CO2. 4P. D. Townsend, P. M. Holliday, D. R. W. Hodgson, and M. J. Cann, unpublished observations. This finding was not unique to Rv1625c as the corresponding mutation in the Class IIIb Slr1991 AC of Synechocystis (K177A) was also responsive to CO2. 4P. D. Townsend, P. M. Holliday, D. R. W. Hodgson, and M. J. Cann, unpublished observations. It is plausible that the substrate determining lysine is not actually a direct site of action for CO2, and we sought evidence for an alternative binding site. Ci has been proposed to help recruit the second metal ion to the active site of the Class IIIb CyaC AC of Spirulina platensis (39Steegborn C. Litvin T.N. Levin L.R. Buck J. Wu H. Nat. Struct. Mol. Biol. 2005; 12: 32-37Crossref PubMed Scopus (138) Google Scholar). Assay of Rv1625c204–443 at varying Mn2+ concentrations revealed that CO2 increased the slope of the dose response (6.6) compared with NaCl (3.0), indicating an increase in cooperativity between binding sites (Fig. 2a). On the basis of their findings in CyaC, Steegborn et al. (39Steegborn C. Litvin T.N. Levin L.R. Buck J. Wu H. Nat. Struct. Mol. Biol. 2005; 12: 32-37Crossref PubMed Scopus (138) Google Scholar) suggested that Ci interacted directly with an active site metal ion. Given our findings on Mn2+ recruitment for Rv1625c, we further investigated this hypothesis. Attempts to identify the metal co-factor as a site of CO2 interaction through enzyme assay proved uninformative, and we, therefore, developed an alternative methodology. Radiolabeled CO2 bound to protein has been previously recovered after mixing and rapid gel filtration (40Lorimer G.H. J. Biol. Chem. 1979; 254: 5599-5601Abstract Full Text PDF PubMed Google Scholar). We, therefore, performed a binding analysis to examine the requirements for CO2 binding to enzyme. CO2 bound Rv1625204–443 with no requirement for metal or substrate (Fig. 2b). Identical results were obtained for the Class IIIb ACs Slr1991 and CyaB1. 4P. D. Townsend, P. M. Holliday, D. R. W. Hodgson, and M. J. Cann, unpublished observations. Control proteins including bovine serum albumin and an alternative hexahistidine-tagged protein 4P. D. Townsend, P. M. Holliday, D. R. W. Hodgson, and M. J. Cann, unpublished observations. showed recovery indistinguishable from buffer alone, indicating an absence of any specific CO2 binding. These data would appear to eliminate a requirement for metal in the active site for CO2 binding, but it is possible that metal co-purified with protein and remained bound to enzyme. We, therefore, investigated CO2 binding in a mutant protein in which both metal binding aspartate residues were mutated to alanine (39Steegborn C. Litvin T.N. Levin L.R. Buck J. Wu H. Nat. Struct. Mol. Biol. 2005; 12: 32-37Crossref PubMed Scopus (138) Google Scholar, 41Tesmer J.J. Sunahara R.K. Johnson R.A. Gosselin G. Gilman A.G. Sprang S.R. Science. 1999; 285: 756-760Crossref PubMed Scopus (279) Google Scholar). The low yield of protein for Rv1625c204–443 D256A/D300A made this experiment impossible for Rv1625c. We, therefore, performed the equivalent experiment in the mutant protein Slr1991120–337 D137A/D181A (Fig. 2c). This confirmed that CO2 binding occurred in the absence of metal despite the fact that the protein was catalytically inactive.4 At physiological pH and CO2 concentrations, only N-terminal α-amino groups and lysine side chain ∊-amino groups are likely to be sufficiently dissociated to react with CO2 (42Lorimer G.H. Trends Biochem. Sci. 1983; 8: 65-68Abstract Full Text PDF Scopus (48) Google Scholar). This is borne out in crystal structures in which carbamates are formed at lysine side chain ∊-amino groups (e.g. Refs. 43Golemi D. Maveyraud L. Vakulenko S. Samama J.P. Mobashery S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14280-14285Crossref PubMed Scopus (200) Google Scholar, 44Hall P.R. Zheng R. Antony L. Pusztai-Carey M. Carey P.R. Yee V.C. EMBO J. 2004; 23: 3621-3631Crossref PubMed Scopus (36) Google Scholar, 45Morollo A.A. Petsko G.A. Ringe D. Biochemistry. 1999; 38: 3293-3301Crossref PubMed Scopus (70) Google Scholar). It is also possible that changes in the local environment may permit arginine to participate in a CO2 binding site (46Cotelesage J.J.H. Puttick J. Goldie H. Rajabi B. Novakovski B. Delbaere L.T.J. Int. J. Biochem. Cell Biol. 2007; 39: 1204-1210Crossref PubMed Scopus (30) Google Scholar). Our findings indicate that the hypothesis that Ci interacts with active site metal is incorrect and that future mechanistic studies should be directed toward sites within the apoprotein. No prokaryotic ACs have been demonstrated to respond in vivo to increases in CO2/HCO3-. This is of obvious importance if prokaryotic ACs are to be posited as sensors of CO2. A demonstration that Rv1625c is responsive to CO2 in vivo is problematic as the numerous ACs in Mycobacterium make specific effects on Rv1625c impossible to distinguish. We, therefore, monitored the activity of Rv1625c expressed in E. coli using a cAMP responsive lacZ reporter construct as a suitable alternative. Using cAMP-driven expression of lacZ as readout, we observed a consistent increase in Rv1625c204–443 activity at elevated CO2 (Fig. 2d). LacZ produced due to endogenous cAMP was not responsive to CO2 (Fig. 2d, Vector). As transcription of the E. coli cya gene (the Class I E. coli AC) is down-regulated by cAMP, expression of Rv1625c204–443 likely reduced endogenous cAMP production and eliminated the possibility that our observations were due to the endogenous Cya AC (47Inada T. Takahashi H. Mizuno T. Aiba H. Mol. Gen. Genet. 1996; 253: 198-204Crossref PubMed Scopus (32) Google Scholar). This demonstrates that a prokaryotic AC can be stimulated by CO2 in an intact bacterium and, thus, fulfils a key criterion for AC as a functional CO2 sensor in bacteria. Building on our findings with Rv1625c, we investigated CO2 as a stimulating ligand for a related mammalian G-protein regulated AC, an experiment of some importance as CO2-stimulated signaling enzymes are not known in eukaryotes (7C1·2C2; Fig. 1a). We investigated the response of 7C1·2C2 to 20 mm total Ci over the pH range 6.5–8.5 (Fig. 3a). Similar to Rv1625c, optimal stimulation of 7C1·2C2 by Ci occurred at low pH, suggesting a direct response to CO2. Assay under conditions of Ci disequilibrium prov" @default.
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• W2142094277 title "Stimulation of Mammalian G-protein-responsive Adenylyl Cyclases by Carbon Dioxide" @default.
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