A Defined Subset of Adenylyl Cyclases Is Regulated by Bicarbonate Ion

Cann, Martin J.; Hammer, Arne; Zhou, Jie; Kanacher, Tobias

doi:10.1074/jbc.m303025200

Cited by 98 publications

(109 citation statements)

References 29 publications

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“…S2 A and B). The sAC/HCO 3 − complex confirms previous evidence that the bicarbonate anion, and not CO 2 , directly stimulates sAC (11,13,29). Soaking apo sAC with biselenite or bisulfite (resolution of 2.05 Å and 1.8 Å, respectively; Tables S1 and S2), both of which structurally resemble HCO 3 − and could be located unambiguously by their anomalous scattering signal (Fig.…”

Section: Resultssupporting

confidence: 63%

“…; Ca 2+ supports substrate binding, and HCO 3 − increases turnover and relieves substrate inhibition (8), and this regulation is conserved in sAC-like enzymes from Cyanobacteria to humans (3,13,29). In a homodimeric, HCO 3 − -regulated sAC homolog from Spirulina platensis, adenylyl cyclase C (CyaC), HCO 3 − appeared to facilitate an active site closure required for catalysis (13), but the HCO 3 − binding site and its mechanism of activation remained unknown.…”

Section: Hco 3 −mentioning

confidence: 99%

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Crystal structures of human soluble adenylyl cyclase reveal mechanisms of catalysis and of its activation through bicarbonate

Kleinboelting

Díaz

Moniot

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

119

215

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cAMP is an evolutionary conserved, prototypic second messenger regulating numerous cellular functions. In mammals, cAMP is synthesized by one of 10 homologous adenylyl cyclases (ACs): nine transmembrane enzymes and one soluble AC (sAC). Among these, only sAC is directly activated by bicarbonate (HCO3−); it thereby serves as a cellular sensor for HCO3−, carbon dioxide (CO2), and pH in physiological functions, such as sperm activation, aqueous humor formation, and metabolic regulation. Here, we describe crystal structures of human sAC catalytic domains in the apo state and in complex with substrate analog, products, and regulators. The activator HCO3− binds adjacent to Arg176, which acts as a switch that enables formation of the catalytic cation sites. An anionic inhibitor, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, inhibits sAC through binding to the active site entrance, which blocks HCO3− activation through steric hindrance and trapping of the Arg176 side chain. Finally, product complexes reveal small, local rearrangements that facilitate catalysis. Our results provide a molecular mechanism for sAC catalysis and cellular HCO3− sensing and a basis for targeting this system with drugs.

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Section: Resultssupporting

confidence: 63%

Section: Hco 3 −mentioning

confidence: 99%

Crystal structures of human soluble adenylyl cyclase reveal mechanisms of catalysis and of its activation through bicarbonate

Kleinboelting

Díaz

Moniot

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

119

215

View full text Add to dashboard Cite

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“…Its absence, along with the observations that bicarbonate (i) does not disrupt microcrystals of free sAC, and (ii) is very specific (Supplementary Table 1 online), yet requires high concentrations (physiological concentrations between 5 and 25 mM) to activate sAC 5,10,24 (corresponding to an estimated off-rate of ~10 6 s −1 , much higher than the turnover rate k cat of ~1 to 4 s −1 ; Supplementary Methods online), might suggest that bicarbonate interacts only very transiently with the enzyme during each catalytic cycle.…”

Section: Potential Bicarbonate Recognition Sitesmentioning

confidence: 99%

“…One difference corresponds to sAC active site residue Thr1139*, which is conserved in the bicarbonate-responsive sACs but replaced by a conserved aspartate in bicarbonate-insensitive bacterial ACs and tmACs 24 . Mutating the analogous threonine to aspartate abolished bicarbonate responsiveness in CyaB1, a cyanobacterial sAC 24 , consistent with Thr1139* contributing to the bicarbonate stimulation of sACs. A potential scenario is that a single bicarbonate molecule could replace the two water molecules that coordinate ion A (Fig.…”

Section: Potential Bicarbonate Recognition Sitesmentioning

confidence: 99%

Bicarbonate activation of adenylyl cyclase via promotion of catalytic active site closure and metal recruitment

Steegborn

Litvin

Levin

et al. 2004

Nat Struct Mol Biol

151

240

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In an evolutionarily conserved signaling pathway, 'soluble' adenylyl cyclases (sACs) synthesize the ubiquitous second messenger cyclic adenosine 3′,5′-monophosphate (cAMP) in response to bicarbonate and calcium signals. Here, we present crystal structures of a cyanobacterial sAC enzyme in complex with ATP analogs, calcium and bicarbonate, which represent distinct catalytic states of the enzyme. The structures reveal that calcium occupies the first ion-binding site and directly mediates nucleotide binding. The single ion-occupied, nucleotide-bound state defines a novel, open adenylyl cyclase state. In contrast, bicarbonate increases the catalytic rate by inducing marked active site closure and recruiting a second, catalytic ion. The phosphates of the bound substrate analogs are rearranged, which would facilitate product formation and release. The mechanisms of calcium and bicarbonate sensing define a reaction pathway involving active site closure and metal recruitment that may be universal for class III cyclases.The ubiquitous second messenger cAMP regulates a large variety of essential physiological processes such as gene expression, chromosome segregation and cellular metabolism. In mammalian cells, cAMP is synthesized by a family of nine transmembrane adenylyl cyclases (tmACs) and one sAC 1 . Unlike tmACs, which localize to the cellular membrane and respond to extracellular stimuli via heterotrimeric G proteins 1 , sAC is found in various intracellular compartments such as the mitochondria and the nucleus 2 . Its localization near intracellular cAMP targets is the impetus for current models of second messenger signal transduction, in which cAMP functions as a locally acting signaling molecule 2-4 .sAC is insensitive to the tmAC regulators calmodulin and heterotrimeric G proteins as well as the nonphysiological activator forskolin; instead, sAC senses physiological levels of bicarbonate 5 . Aside from its role as a universal physiological buffer maintaining cellular and extracellular pH, bicarbonate functions as a signaling molecule 3 , regulating many biological processes in mammals such as fertility 6 , acid-base homeostasis, breathing rate, metabolism and fluid transport (reviewed in ref. 7). As the only known signaling enzyme sensitive to physiological fluctuations of bicarbonate 5 , sAC probably mediates each of these processes. Bicarbonate activation of sAC is essential for sperm motility 8 as well as for pH-dependent COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. acid secretion in the epididymis and possibly the kidney 9 . In addition to its bicarbonate sensitivity, sAC is synergistically activated by calcium 10 , and this potentiation seems to be important for sperm maturation 11 . NIH Public AccessPrevious work has revealed the overall structure of tmAC enzymes and suggested a twometal ion mechanism for catalysis 12,13 . Despite their different regulation, mammalian sAC and tmACs are grouped into the nucleotidyl cyclase class III based on sequ...

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“…Mammalian sAC (Chen et al 2000;Litvin et al 2003) and a subset of the bacterial sAC-like ACs (Cann et al 2003) are directly stimulated by bicarbonate anions. The mechanism of bicarbonate stimulation is conserved between bacterial and mammalian enzymes.…”

Section: Introductionmentioning

confidence: 99%

Conservation of functional domain structure in bicarbonate-regulated ?soluble? adenylyl cyclases in bacteria and eukaryotes

2004

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Soluble adenylyl cyclase (sAC) is an evolutionarily conserved bicarbonate sensor. In mammals, it is responsible for bicarbonate-induced, cAMP-dependent processes in sperm required for fertilization and postulated to be involved in other bicarbonate-and carbon dioxide-dependent functions throughout the body. Among eukaryotes, sAC-like cyclases have been detected in mammals and in the fungi Dictyostelium; these enzymes display extensive similarity extending through two cyclase catalytic domains and a long carboxy terminal extension. sAC-like cyclases are also found in a number of bacterial phyla (Cyanobacteria, Actinobacteria, and Proteobacteria), but these enzymes generally possess only a single catalytic domain and little, if any, homology with the remainder of the mammalian protein. Database mining through a number of recently sequenced genomes identified sAC orthologues in additional metazoan phyla (Arthropoda and Chordata) and additional bacterial phyla (Chloroflexi). Interestingly, the Chloroflexi sAC-like cyclases, a family of three enzymes from the thermophilic eubacterium, Chloroflexus aurantiacus, are more similar to eukaryotic sAC-like cyclases (i.e., mammalian sAC and Dictyostelium SgcA) than they are to other bacterial adenylyl cyclases (ACs) (i.e., from Cyanobacteria). The Chloroflexus sAC-like cyclases each possess two cyclase catalytic domains and extensive similarity with mammalian enzymes through their carboxy termini. We cloned one of the Chloroflexus sAC-like cyclases and confirmed it to be stimulated by bicarbonate. These data extend the family of organisms possessing bicarbonate-responsive ACs to numerous phyla within the bacterial and eukaryotic kingdoms.

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A Defined Subset of Adenylyl Cyclases Is Regulated by Bicarbonate Ion

Cited by 98 publications

References 29 publications

Crystal structures of human soluble adenylyl cyclase reveal mechanisms of catalysis and of its activation through bicarbonate

Crystal structures of human soluble adenylyl cyclase reveal mechanisms of catalysis and of its activation through bicarbonate

Bicarbonate activation of adenylyl cyclase via promotion of catalytic active site closure and metal recruitment

Conservation of functional domain structure in bicarbonate-regulated ?soluble? adenylyl cyclases in bacteria and eukaryotes

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