A guanylyl cyclase from Paramecium with 22 transmembrane spans. Expression of the catalytic domains and formation of chimeras with the catalytic domains of mammalian adenylyl cyclases

Paramecium has a 280-kDa guanylyl cyclase. The N terminus resembles a P-type ATPase, and the C terminus is a guanylyl cyclase with the membrane topology of canonical mammalian adenylyl cyclases, yet with the cytosolic loops, C1 and C2, inverted compared with the mammalian order. We expressed in Escherichia coli the cytoplasmic domains of the protozoan guanylyl cyclase, independently and linked by a peptide, as soluble proteins. The His(6)-tagged proteins were enriched by affinity chromatography and analyzed by immunoblotting. Guanylyl cyclase activity was reconstituted upon mixing of the recombinant C1a- and C2-positioned domains and in a linked C1a-C2 construct. Adenylyl cyclase activity was minimal. The nucleotide substrate specificity was switched from GTP to ATP upon mutation of the substrate defining amino acids Glu(1681) and Ser(1748) in the C1-positioned domain to the adenylyl cyclase specific amino acids Lys and Asp. Using the C2 domains of mammalian adenylyl cyclases type II or IX and the C2-positioned domain from the Paramecium guanylyl cyclase we reconstituted a soluble, all C2 adenylyl cyclase. All enzymes containing protozoan domains were not affected by Galpha(s)/GTP or forskolin, and P site inhibitors were only slightly effective.     

CyaG, a novel cyanobacterial adenylyl cyclase and a possible ancestor of mammalian guanylyl cyclases

A novel gene encoding an adenylyl cyclase, designated cyaG, was identified in the filamentous cyanobacterium Spirulina platensis. The predicted amino acid sequence of the C-terminal region of cyaG was similar to the catalytic domains of Class III adenylyl and guanylyl cyclases. The N-terminal region next to the catalytic domain of CyaG was similar to the dimerization domain, which is highly conserved among guanylyl cyclases. As a whole, CyaG is more closely related to guanylyl cyclases than to adenylyl cyclases in its primary structure. The catalytic domain of CyaG was expressed in Escherichia coli and partially purified. CyaG showed adenylyl cyclase (but not guanylyl cyclase) activity. By site-directed mutagenesis of three amino acid residues (Lys(533), Ile(603), and Asp(605)) within the purine ring recognition site of CyaG to Glu, Arg, and Cys, respectively, CyaG was transformed to a guanylyl cyclase that produced cGMP instead of cAMP. Thus having properties of both cyclases, CyaG may therefore represent a critical position in the evolution of Class III adenylyl and guanylyl cyclases.     

Adenylyl cyclase Rv0386 from Mycobacterium tuberculosis H37Rv uses a novel mode for substrate selection

Class III adenylyl cyclases usually possess six highly conserved catalytic residues. Deviations in these canonical amino acids are observed in several putative adenylyl cyclase genes as apparent in several bacterial genomes. This suggests that a variety of catalytic mechanisms may actually exist. The gene Rv0386 from Mycobacterium tuberculosis codes for an adenylyl cyclase catalytic domain fused to an AAA-ATPase and a helix-turn-helix DNA-binding domain. In Rv0386, the standard substrate, adenine-defining lysine-aspartate couple is replaced by glutamine-asparagine. The recombinant adenylyl cyclase domain was active with a V(max) of 8 nmol cAMP.mg(-1).min(-1). Unusual for adenylyl cyclases, Rv0386 displayed 20% guanylyl cyclase side-activity with GTP as a substrate. Mutation of the glutamine-asparagine pair either to alanine residues or to the canonical lysine-aspartate consensus abolished activity. This argues for a novel mechanism of substrate selection which depends on two non-canonical residues. Data from individual and coordinated point mutations suggest a model for purine definition based on an amide switch related to that previously identified in cyclic nucleotide phosphodiesterases.     

GTPgammaS regulation of a 12-transmembrane guanylyl cyclase is retained after mutation to an adenylyl cyclase

DdGCA is a Dictyostelium guanylyl cyclase with a topology typical for mammalian adenylyl cyclases containing 12 transmembrane-spanning regions and two cyclase domain. In Dictyostelium cells heterotrimeric G-proteins are essential for guanylyl cyclase activation by extracellular cAMP. In lysates, guanylyl cyclase activity is strongly stimulated by guanosine 5'-3-O-(thio) triphosphate (GTPgammaS), which is also a substrate of the enzyme. DdGCA was converted to an adenylyl cyclase by introducing three point mutations. Expression of the obtained DdGCA(kqd) in adenylyl cyclase-defective cells restored the phenotype of the mutant. GTPgammaS stimulated the adenylyl cyclase activity of DdGCA(kqd) with properties similar to those of the wild-type enzyme (decrease of K(m) and increase of V(max)), demonstrating that GTPgammaS stimulation is independent of substrate specificity. Furthermore, GTPgammaS activation of DdGCA(kqd) is retained in several null mutants of Galpha and Gbeta proteins, indicating that GTPgammaS activation is not mediated by a heterotrimeric G-protein but possibly by a monomeric G-protein.     

Studying the structure and regulation of soluble guanylyl cyclase

Soluble guanylyl cyclase acts as the receptor for the signaling molecule nitric oxide. The enzyme consists of two different subunits. Each subunit shows the cyclase catalytic domain, which is also conserved in the membrane-bound guanylyl cyclases and the adenylyl cyclases. The N-terminal regions of the subunits are responsible for binding of the prosthetic heme group of the enzyme, which is required for the stimulatory effect of nitric oxide (NO). The five-coordinated ferrous heme displays a histidine as the axial ligand; activation of soluble guanylyl cyclase by NO is initiated by binding of NO to the heme iron and proceeds via breaking of the histidine-to-iron bond. Recently, a novel pharmacological and possibly physiological principle of guanylyl cyclase sensitization was demonstrated. The substance YC-1 has been shown to activate the enzyme independent of NO, to potentiate the effect of submaximally effective NO concentrations, and to turn carbon monoxide into an effective activator of soluble guanylyl cyclase.     

Guanylyl cyclase activity associated with putative bifunctional integral membrane proteins in Plasmodium falciparum

We report here that guanylyl cyclase activity is associated with two large integral membrane proteins (PfGCalpha and PfGCbeta) in the human malaria parasite Plasmodium falciparum. Unusually, the proteins appear to be bifunctional; their amino-terminal regions have strong similarity with P-type ATPases, and the sequence and structure of the carboxyl-terminal regions conform to that of G protein-dependent adenylyl cyclases, with two sets of six transmembrane sequences, each followed by a catalytic domain (C1 and C2). However, amino acids that are enzymatically important and present in the C2 domain of mammalian adenylyl cyclases are located in the C1 domain of the P. falciparum proteins and vice versa. In addition, certain key residues in these domains are more characteristic of guanylyl cyclases. Consistent with this, guanylyl cyclase activity was obtained following expression of the catalytic domains of PfGCbeta in Escherichia coli. In P. falciparum, expression of both genes was detectable in the sexual but not the asexual blood stages of the life cycle, and PfGCalpha was localized to the parasite/parasitophorous vacuole membrane region of gametocytes. The profound structural differences identified between mammalian and parasite guanylyl cyclases suggest that aspects of this signaling pathway may be mechanistically distinct.     

The primary structure of the larger subunit of soluble guanylyl cyclase from bovine lung. Homology between the two subunits of the enzyme

The primary structure of the larger subunit of the soluble guanylyl cyclase from bovine lung, which catalyzes the formation of cyclic GMP from GTP, has been determined. Two clones, isolated from two bovine libraries yielded a total of 3261 bp with a coding region of 2073 bp. The open reading frame encodes a protein of 691 amino acids and a molecular mass of 77,500. The deduced amino acid sequence reveals regions which are, to a large extent, homologous to the sequence of the smaller subunit of the enzyme as well as to the sequences of other gyanylyl and adenylyl to a large extent, homologous to the sequence of the smaller subunit of the enzyme as well as to the sequences of other gyanylyl and adenylyl cyclases.     

The adenylyl and guanylyl cyclase superfamily

New structures solved in 1997 revealed that the adenylyl cyclase core consists of a pair of catalytic domains arranged in a wreath. Homologous catalytic domains are arranged in diverse adenylyl and guanylyl cyclases as symmetric homodimers or pseudosymmetric heterodimers. The kinship of the adenylyl and guanylyl cyclases has been confirmed by the structure-based interconversion of their nucleotide specificities. Catalysis is activated when two metal-binding aspartate residues on one domain are juxtaposed with a key aspargine—arginine pair on the other. Allosteric activators of mammalian adenylyl cyclase, forskolin and the stimulatory G protein α subunit, promote the catalytically optimal juxtaposition of the two domains.     

Sequence of a human brain adenylyl cyclase partial cDNA: evidence for a consensus cyclase specific domain.

A cDNA coding for a human brain adenylyl cyclase was isolated and sequenced. The deduced partial 675 amino-acid sequence was compared with those of other known adenylyl and guanylyl cyclases. Comparison of this predicted amino-acid sequence with that of bovine brain (type I) and rat olfactory (type III) adenylyl cyclase indicated a significant homology with the carboxyl-terminal halves of both enzymes. The homology between the human adenylyl cyclase and the other two mammalian adenylyl cyclase also appears at the topographic level. Indeed, the human enzyme includes a extremely hydrophobic region containing six potential membrane-spanning segments followed by a large hydrophilic domain. At the beginning of the hydrophilic domain, there is a 250 amino-acid region which shows not only a striking homology with the bovine and rat adenylyl cyclase (86% of similarity and 57% of identity), but also a significant homology with non-mammalian adenylyl cyclase and guanylyl cyclases. We found that this 250 amino-acid domain contains a sequence of about 165 amino-acids which is highly conserved in most of the known nucleotide cyclases suggesting that it includes residues that are critical for the function of the enzymes.     

A functional chimera of mammalian guanylyl and adenylyl cyclases

Adenylyl and guanylyl cyclases synthesize second messenger molecules by intramolecular esterification of purine nucleotides, i.e., cAMP from ATP and cGMP from GTP, respectively. Despite their sequence homology, both families of mammalian cyclases show remarkably different regulatory patterns. In an attempt to define the functional domains in adenylyl cyclase responsible for their isotypic-common activation by Galphas or forskolin, dimeric chimeras were constructed from soluble guanylyl cyclase alpha1 subunit and the C-terminal halves of adenylyl cyclases type I, II, or V. The cyclase-hybrid generated cAMP and was inhibited by P-site ligands. The data establish structural equivalence and the ability of functional complement at the catalytic sites in both cyclases. Detailed enzymatic characterization of the chimeric cyclase revealed a crucial role of the N-terminal adenylyl cyclase half for stimulatory actions, and a major importance of the C-terminal part for nucleotide specificity.     

Mutational analysis of the Mycobacterium tuberculosis Rv1625c adenylyl cyclase: residues that confer nucleotide specificity contribute to dimerization

The mycobacterial Rv1625c gene product is an adenylyl cyclase with sequence similarity to the mammalian enzymes. The catalytic domain of the enzyme forms a homodimer and residues specifying adenosine triphosphate (ATP) specificity lie at the dimer interface. Mutation of these residues to those present in guanylyl cyclases failed to convert the enzyme to a guanylyl cyclase, but dramatically reduced its adenylyl cyclase activity and altered its oligomeric state. Computational modeling revealed subtle differences in the dimer interface that could explain the biochemical data, suggesting that the structural and catalytic features of this homodimeric adenylyl cyclase are in contrast to those of the heterodimeric mammalian enzymes.     

A novel mechanism for adenylyl cyclase inhibition from the crystal structure of its complex with catechol estrogen

Catechol estrogens are steroid metabolites that elicit physiological responses through binding to a variety of cellular targets. We show here that catechol estrogens directly inhibit soluble adenylyl cyclases and the abundant trans-membrane adenylyl cyclases. Catechol estrogen inhibition is non-competitive with respect to the substrate ATP, and we solved the crystal structure of a catechol estrogen bound to a soluble adenylyl cyclase from Spirulina platensis in complex with a substrate analog. The catechol estrogen is bound to a newly identified, conserved hydrophobic patch near the active center but distinct from the ATP-binding cleft. Inhibitor binding leads to a chelating interaction between the catechol estrogen hydroxyl groups and the catalytic magnesium ion, distorting the active site and trapping the enzyme substrate complex in a non-productive conformation. This novel inhibition mechanism likely applies to other adenylyl cyclase inhibitors, and the identified ligand-binding site has important implications for the development of specific adenylyl cyclase inhibitors.     

Class III nucleotide cyclases in bacteria and archaebacteria: lineage-specific expansion of adenylyl cyclases and a dearth of guanylyl cyclases

The Class III nucleotide cyclases are found in bacteria, eukaryotes and archaebacteria. Our survey of the bacterial and archaebacterial genome and plasmid sequences identified 193 Class III cyclase genes in only 29 species, of which we predict the majority to be adenylyl cyclases. Interestingly, several putative cyclase genes were found to have non-conserved substrate specifying residues. Ancestors of the eukaryotic C1-C2 domain containing soluble adenylyl cyclases as well as the protist guanylyl cyclases were found in bacteria. Diverse domains were fused to the cyclase domain and phylogenetic analysis indicated that most proteins within a single cluster have similar domain compositions, emphasising the ancient evolutionary origin and versatility of the cyclase domain.     

Sequence homologies between guanylyl cyclases and structural analogies to other signal-transducing proteins

The cyclic GMP-forming enzyme guanylyl cyclase exists in cytosolic and in membrane-bound forms differing in structure and regulations. Determination of the primary structures of the guanylyl cyclases revealed that the cytosolic enzyme form consists of two similar subunits and that membrane-bound guanylyl cyclases represent enzyme forms in which the catalytic part is located in an intracellular, C-terminal domain and is regulated by an extracelluar, N-terminal receptor domain. A domain of 250 amino acids conserved in all guanylyl cyclases appears to be required for the formation of cyclic nucleotide, as this homologous domain is also found in the cytosolic regions of the adenylyl cyclase. The general structures of guanylyl cyclases shows similarities with other signal transducing enzymes such as protein-tyrosine phosphatases and protein-tyrosine kinases. which also exist in cytosolic and receptor-linked forms.     

Guanylyl cyclases in unicellular organisms

Guanylyl cyclases in eukaryotic unicells were biochemically investigated in the ciliates Paramecium and Tetrahymena, in the malaria parasite Plasmodium and in the ameboid Dictyostelium. In ciliates guanylyl cyclase activity is calcium-regulated suggesting a structural kinship to similarly regulated membrane-bound guanylyl cyclases in vertebrates. Yet, cloning of ciliate guanylyl cyclases revealed a novel combination of known modular building blocks. Two cyclase homology domains are inversely arranged in a topology of mammalian adenylyl cyclases, containing two cassettes of six transmembrane spans. In addition the protozoan guanylyl cyclases contain an N-terminal P-type ATPase-like domain. Sequence comparisons indicate a compromised ATPase function. The adopted novel function remains enigmatic to date. The topology of the guanylyl cyclase domain in all protozoans investigated is identical. A recently identified Dictyostelium guanylyl cyclase lacks the N-terminal P-type ATPase domain. The close functional relation of Paramecium guanylyl cyclases to mammalian adenylyl cyclases has been established by heterologous expression, respective point mutations and a series of active mammalian adenylyl cyclase/Paramecium guanylyl cyclase chimeras. The unique structure of protozoan guanylyl cyclases suggests that unexpectedly they do not share a common guanylyl cyclase ancestor with their vertebrate congeners but probably originated from an ancestral mammalian-type adenylyl cyclase.     

The Dictyostelium homologue of mammalian soluble adenylyl cyclase encodes a guanylyl cyclase

A new Dictyostelium discoideum cyclase gene was identified that encodes a protein (sGC) with 35% similarity to mammalian soluble adenylyl cyclase (sAC). Gene disruption of sGC has no effect on adenylyl cyclase activity and results in a >10-fold reduction in guanylyl cyclase activity. The scg- null mutants show reduced chemotactic sensitivity and aggregate poorly under stringent conditions. With Mn(2+)/GTP as substrate, most of the sGC activity is soluble, but with the more physiological Mg(2+)/GTP the activity is detected in membranes and stimulated by GTPgammaS. Unexpectedly, orthologues of sGC and sAC are present in bacteria and vertebrates, but absent from Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana and Saccharomyces cerevisiae.     

A structural basis for the role of nucleotide specifying residues in regulating the oligomerization of the Rv1625c adenylyl cyclase from M. tuberculosis

The Rv1625c Class III adenylyl cyclase from Mycobacterium tuberculosis is a homodimeric enzyme with two catalytic centers at the dimer interface, and shows sequence similarity with the mammalian adenylyl and guanylyl cyclases. Mutation of the substrate-specifying residues in the catalytic domain of Rv1625c, either independently or together, to those present in guanylyl cyclases not only failed to confer guanylyl cyclase activity to the protein, but also severely abrogated the adenylyl cyclase activity of the enzyme. Biochemical analysis revealed alterations in the behavior of the mutants on ion-exchange chromatography, indicating differences in the surface-exposed charge upon mutation of substrate-specifying residues. The mutant proteins showed alterations in oligomeric status as compared to the wild-type enzyme, and differing abilities to heterodimerize with the wild-type protein. The crystal structure of a mutant has been solved to a resolution of 2.7A. On the basis of the structure, and additional biochemical studies, we provide possible reasons for the altered properties of the mutant proteins, as well as highlight unique structural features of the Rv1625c adenylyl cyclase.     

Activation of soluble guanylyl cyclase at the leading edge during Dictyostelium chemotaxis

Dictyostelium contains two guanylyl cyclases, GCA, a 12-transmembrane enzyme, and sGC, a homologue of mammalian soluble adenylyl cyclase. sGC provides nearly all chemoattractant-stimulated cGMP formation and is essential for efficient chemotaxis toward cAMP. We show that in resting cells the major fraction of the sGC-GFP fusion protein localizes to the cytosol, and a small fraction is associated to the cell cortex. With the artificial substrate Mn2+/GTP, sGC activity and protein exhibit a similar distribution between soluble and particulate fraction of cell lysates. However, with the physiological substrate Mg2+/GTP, sGC in the cytosol is nearly inactive, whereas the particulate enzyme shows high enzyme activity. Reconstitution experiments reveal that inactive cytosolic sGC acquires catalytic activity with Mg2+/GTP upon association to the membrane. Stimulation of cells with cAMP results in a twofold increase of membrane-localized sGC-GFP, which is accompanied by an increase of the membrane-associated guanylyl cyclase activity. In a cAMP gradient, sGC-GFP localizes to the anterior cell cortex, suggesting that in chemotacting cells, sGC is activated at the leading edge of the cell.     

The adenylyl cyclase family

Hormone-sensitive adenylyl cyclase is a model system for the study of receptor-mediated signal transduction. It is comprised of three types of components: 1) receptors for hormones that regulate cyclic AMP (cAMP) synthesis, 2) regulatory GTP binding proteins (G proteins), and 3) the family of enzymes, the adenylyl cyclases. Concentrations of cAMP are altered by at least 35 different stimulatory or inhibitory hormones and neurotransmitters. Other signalling pathways may also influence cAMP production through regulation of particular adenylyl cyclase subtypes. The second messenger, cAMP propagates the hormone signal through the effects of cAMP-dependent protein kinase.While structural information on the adenylyl cyclases is limited, a cDNA clone for a calmodulin-sensitive form of bovine brain adenylyl cyclase has been isolated. The amino acid sequence encoded by the Type I cDNA is approximately 40% identical to those specified by three other adenylyl cyclase cDNAs that have been cloned subsequently. This degree of structural variation implies that there must be functional differences between the adenylyl cyclases.