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.     

Molecular basis for P-site inhibition of adenylyl cyclase

P-site inhibitors are adenosine and adenine nucleotide analogues that inhibit adenylyl cyclase, the effector enzyme that catalyzes the synthesis of cyclic AMP from ATP. Some of these inhibitors may represent physiological regulators of adenylyl cyclase, and the most potent may ultimately serve as useful therapeutic agents. Described here are crystal structures of the catalytic core of adenylyl cyclase complexed with two such P-site inhibitors, 2'-deoxyadenosine 3'-monophosphate (2'-d-3'-AMP) and 2',5'-dideoxyadenosine 3'-triphosphate (2',5'-dd-3'-ATP). Both inhibitors bind in the active site yet exhibit non- or uncompetitive patterns of inhibition. While most P-site inhibitors require pyrophosphate (PP(i)) as a coinhibitor, 2',5'-dd-3'-ATP is a potent inhibitor by itself. The crystal structure reveals that this inhibitor exhibits two binding modes: one with the nucleoside moiety bound to the nucleoside binding pocket of the enzyme and the other with the beta and gamma phosphates bound to the pyrophosphate site of the 2'-d-3'-AMP.PP(i) complex. A single metal binding site is observed in the complex with 2'-d-3'-AMP, whereas two are observed in the complex with 2', 5'-dd-3'-ATP. Even though P-site inhibitors are typically 10 times more potent in the presence of Mn(2+), the electron density maps reveal no inherent preference of either metal site for Mn(2+) over Mg(2+). 2',5'-dd-3'-ATP binds to the catalytic core of adenylyl cyclase with a K(d) of 2.4 microM in the presence of Mg(2+) and 0.2 microM in the presence of Mn(2+). Pyrophosphate does not compete with 2',5'-dd-3'-ATP and enhances inhibition.     

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

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.     

Origin of asymmetry in adenylyl cyclases: structures of Mycobacterium tuberculosis Rv1900c

Rv1900c, a Mycobacterium tuberculosis adenylyl cyclase, is composed of an N-terminal alpha/beta-hydrolase domain and a C-terminal cyclase homology domain. It has an unusual 7% guanylyl cyclase side-activity. A canonical substrate-defining lysine and a catalytic asparagine indispensable for mammalian adenylyl cyclase activity correspond to N342 and H402 in Rv1900c. Mutagenic analysis indicates that these residues are dispensable for activity of Rv1900c. Structures of the cyclase homology domain, solved to 2.4 A both with and without an ATP analog, form isologous, but asymmetric homodimers. The noncanonical N342 and H402 do not interact with the substrate. Subunits of the unliganded open dimer move substantially upon binding substrate, forming a closed dimer similar to the mammalian cyclase heterodimers, in which one interfacial active site is occupied and the quasi-dyad-related active site is occluded. This asymmetry indicates that both active sites cannot simultaneously be catalytically active. Such a mechanism of half-of-sites-reactivity suggests that mammalian heterodimeric adenylyl cyclases may have evolved from gene duplication of a primitive prokaryote-type cyclase, followed by loss of function in one active site.     

Inhibition of the soluble form of testis adenylate cyclase by catechol estrogens and other catechols

The soluble form of rat germ cell adenylate cyclase was inhibited by compounds with a catechol moiety. Among the naturally occurring catechols tested, catechol estrogens were the most potent inhibitors. Catechol estrogens at 2-6 microM inhibited enzyme activity by 50% and almost completely at 30-100 microM concentration. The inhibitory activity of catechol estrogens depends on the catechol moiety of the molecule. Catechol per se also inhibited the activity of this enzyme, 50% inhibition being achieved at about 11 microM. The two hydroxyls of the catechol moiety are essential for the inhibitory interaction with the enzyme. Thus, aromatic compounds containing only one hydroxyl group in the benzene ring, such as tyrosine, phenylephrine, estradiol, and 6 alpha-hydroxyestradiol were either completely inactive or had marginal inhibitory activity at concentrations up to 0.3-1 mM. Moreover, methylation of the hydroxyl groups of the catechol moiety of the catechol estrogens as in 2-methoxyestradiol 3-methyl ether rendered the catechol estrogens inactive. The inhibitory potency of these compounds varied greatly depending on the structure associated with the catechol ring. Thus, compounds in which catechol is associated with an aliphatic side chain, such as dopamine, L-dopa, norepinephrine, and isoproterenol, were about 11- to 34-fold less potent than catechol. On the other hand, compounds in which catechol is associated either with a hydroaromatic ring system, as in apomorphine, or with an alicyclic ring system, as in catechol estrogens, were about 2- to 5-fold more potent than catechol. The inhibitory effect of dopamine, apomorphine, and catechol estrogens was not affected by specific D-1 or D-2 antagonist, indicating that they do not act via receptors for dopamine.     

Biochemical and computational insights into the anti-aromatase activity of natural catechol estrogens

High levels of endogenous estrogens are associated with increased risks of breast cancer. Estrogen levels are mainly increased by the activity of the aromatase enzyme and reduced by oxidative/conjugative metabolic pathways. In this paper, we demonstrate for the first time that catechol estrogen metabolites are potent aromatase inhibitors, thus establishing a link between aromatase activity and the processes involved in estrogen metabolism. In particular, the anti-aromatase activity of a set of natural hydroxyl and methoxyl estrogen metabolites was investigated using biochemical methods and subsequently compared with the anti-aromatase potency of estradiol and two reference aromatase inhibitors. Catechol estrogens proved to be strong inhibitors with an anti-aromatase potency two orders of magnitude higher than estradiol. A competitive inhibition mechanism was found for the most potent molecule, 2-hydroxyestradiol (2-OHE(2)) and a rational model identifying the interaction determinants of the metabolites with the enzyme is proposed based on ab initio quantum-mechanical calculations. A strong relationship between activity and electrostatic properties was found for catechol estrogens. Moreover, our results suggest that natural catechol estrogens may be involved in the control mechanisms of estrogen production.     

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.     

Inhibition of Bordetella pertussis and Bacillus anthracis adenylyl cyclases by polyadenylate and "P"-site agonists

Inhibition of adenylyl cyclases from Bacillus anthrasis and Bordetella pertussis by polyadenylate and by the most potent "P"-site agonists was investigated. These bacterial adenylyl cyclases differed in their sensitivity to inhibition by nominal "P"-site agents and in the effect of divalent cations on this inhibition. The enzyme from Bordetella pertussis was relatively insensitive to inhibition by "P"-site agonists, exhibiting a rank order of potency of 2'd3'AMP greater than 3'-AMP greater than 2',5'-ddAdo approximately Ado approximately 2'-dAdo, with IC50 values for 2'd3'AMP and 3'-AMP of 1-3 mM. Inhibition by 2'd3'AMP, however, was not affected by divalent cation, making it distinct from "P"-site-mediated inhibition of most mammalian adenylyl cyclases. The sensitivity to these nucleosides was comparable with potency for inhibition of bovine sperm adenylyl cyclase but was 3 orders of magnitude less potent than for activated enzyme from bovine or rat brain. The Bordetella pertussis enzyme was similarly insensitive to inhibition by polyadenylate, with 16 microM inhibiting less than 20%. By comparison, Bacillus anthrasis adenylyl cyclase was more potently inhibited by 2'd3'AMP (IC50 approximately 85 microM) but not by the other nucleosides (less than 15% inhibition at 1 mM), and inhibition by 2'd3'AMP was optimally enhanced by 5-10 mM Mg2+ or Mn2+, as is typical for inhibition by "P"-site agonists. The Bacillus anthrasis enzyme was potently inhibited by polyadenylate (IC50 approximately 0.3 microM), comparable to inhibition of brain adenylyl cyclases. Sensitivity of Bacillus anthrasis adenylyl cyclase to poly(A) was diminished somewhat by Ca2+/calmodulin (to IC50 approximately 1 microM) although Ca2+/calmodulin was without effect on inhibition by 2'd3'AMP. In contrast to inhibition of mammalian adenylyl cyclases via the "P"-site, inhibition of both bacterial adenylyl cyclases by 2'd3'AMP was competitive with respect to substrate MgATP. The data indicate basic differences in susceptibilities of these bacterial adenylyl cyclases to inhibition by poly(A), by adenosine analogs, and the effects of divalent cations. Although the potency of 2'd3'AMP and the metal-dependent nature of inhibition of Bacillus anthrasis adenylyl cyclase shared characteristics of "P"-site-mediated inhibition, the fact that inhibition of both bacterial adenylyl cyclases was competitive with respect to substrate strongly suggests that this inhibition was at the catalytic site and that these bacterial enzymes do not contain a distinct "P"-site.     

Inhibition by calcium of mammalian adenylyl cyclases

Ca(2+) regulates mammalian adenylyl cyclases in a type-specific manner. Stimulatory regulation is moderately well understood. By contrast, even the concentration range over which Ca(2+) inhibits adenylyl cyclases AC5 and AC6 is not unambiguously defined; even less so is the mechanism of inhibition. In the present study, we compared the regulation of Ca(2+)-stimulable and Ca(2+)-inhibitable adenylyl cyclases expressed in Sf9 cells with tissues that predominantly express these activities in the mouse brain. Soluble forms of AC5 containing either intact or truncated major cytosolic domains were also examined. All adenylyl cyclases, except AC2 and the soluble forms of AC5, displayed biphasic Ca(2+) responses, suggesting the presence of two Ca(2+) sites of high ( approximately 0.2 microM) and low affinity ( approximately 0.1 mM). With a high affinity, Ca(2+) (i) stimulated AC1 and cerebellar adenylyl cyclases, (ii) inhibited AC6 and striatal adenylyl cyclase, and (iii) was without effect on AC2. With a low affinity, Ca(2+) inhibited all adenylyl cyclases, including AC1, AC2, AC6, and both soluble forms of AC5. The mechanism of both high and low affinity inhibition was revealed to be competition for a stimulatory Mg(2+) site(s). A remarkable selectivity for Ca(2+) was displayed by the high affinity site, with a K(i) value of approximately 0.2 microM, in the face of a 5000-fold excess of Mg(2+). The present results show that high and low affinity inhibition by Ca(2+) can be clearly distinguished and that the inhibition occurs type-specifically in discrete adenylyl cyclases. Distinction between these sites is essential, or quite spurious inferences may be drawn on the nature or location of high affinity binding sites in the Ca(2+)-inhibitable adenylyl cyclases.     

Hydroxamate based inhibitors of adenylyl cyclase. Part 1: the effect of acyclic linkers on P-site binding

The adenylyl cyclases (ACs) are a family of enzymes that are key elements of signal transduction by virtue of their ability to convert ATP to cAMP. The catalytic mechanism of this transformation proceeds through initial binding of ATP to the purine binding site (P-site) followed by metal mediated cyclization with loss of pyrophosphate. Crystallographic analysis of ACs with known inhibitors reveals the presence of two metals in the active site. Presently, nine isoforms of adenylyl cyclase are known and unique isoform combinations are expressed in a tissue specific manner. The development of isoform specific inhibitors of adenylyl cyclase may prove to be a useful strategy toward the design of novel therapeutic agents. In order to develop novel AC inhibitors, we have chosen a design approach utilizing molecules with the adenine ring system joined to a metal-coordinating hydroxamic acid via flexible acyclic linkers. The designed inhibitors were assayed against type V AC with the size and heteroatom content of the linkers varied to probe the interaction of the nucleotide and metal binding sites within the enzyme.     

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.     

Conversion of a guanylyl cyclase to an adenylyl cyclase

Guanylyl cyclases catalyze the formation of cGMP from GTP, but display extensive identity at the catalytic domain primary amino acid level with the adenylyl cyclases. The recent solving of the crystal structures of soluble forms of adenylyl cyclase has resulted in predictions of those amino acids important for substrate specificity. Modeling of a membrane-bound homodimeric guanylyl cyclase predicted the comparable amino acids that would interact with the guanine ring. Based on these structural data, the replacement of three key residues in the heterodimeric form of soluble guanylyl cyclase has led to a complete conversion in substrate specificity. Furthermore, the mutant enzyme remained fully sensitive to sodium nitroprusside, a nitric oxide donor.     

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.     

Enzymatic synthesis of unlabeled and beta-(32)P-labeled beta-L-2', 3'-dideoxyadenosine-5'-triphosphate as a potent inhibitor of adenylyl cyclases and its use as reversible binding ligand

beta-L-2',3'-Dideoxyadenosine-5'-triphosphate (beta-L-2', 3'-dd-5'-ATP) was prepared enzymatically from the corresponding monophosphate by the use of adenylate kinase, creatine phosphate, and creatine kinase in a single step. The beta-(32)P-labeled analog was prepared similarly, but in a two step reaction. beta-L-2', 3'-dd-5'-ATP inhibited adenylyl cyclase from rat brain competitively with respect to substrate (5'-ATP.Mn(2+)) and exhibited an IC(50) approximately 24 nM. The labeled ligand was used in the development of a reversible binding assay for adenylyl cyclases. Binding of beta-L-2',3'-dd-[beta-(32)P]5'-ATP was saturable with increasing concentrations of ligand and increased in proportion to membrane protein, and was enhanced by Mn(2+) to a greater extent than by Mg(2+). Binding was displaced with adenine nucleotides known to be either competitive or noncompetitive inhibitors but not by agents known not to act on the cyclase, or by 3-isobutyl-1-methylxanthine, creatine phosphate, or creatine kinase. Binding was rapid, with a half-time for the on-rate <1.8 min and for the off-rate <0.8 min. The potency and mechanism of the inhibition of this ligand and the pattern of agents that displace binding suggest an interaction with adenylyl cyclase per se and to a configuration of the enzyme consistent with an interaction at the catalytic active site. The data suggest that this is a pretransition state inhibitor and contrasts with the equipotent 2',5'-dd-3'ATP, a post-transition state noncompetitive inhibitor.     

Structure-based inhibitor discovery against adenylyl cyclase toxins from pathogenic bacteria that cause anthrax and whooping cough

Edema factor (EF) and CyaA are adenylyl cyclase toxins secreted by pathogenic bacteria that cause anthrax and whooping cough, respectively. Using the structure of the catalytic site of EF, we screened a data base of commercially available, small molecular weight chemicals for those that could specifically inhibit adenylyl cyclase activity of EF. From 24 compounds tested, we have identified one quinazoline compound, ethyl 5-aminopyrazolo[1,5-a]quinazoline-3-carboxylate, that specifically inhibits adenylyl cyclase activity of EF and CyaA with approximately 20 microm Ki. This compound neither affects the activity of host resident adenylyl cyclases type I, II, and V nor exhibits promiscuous inhibition. The compound is a competitive inhibitor, consistent with the prediction that it binds to the adenine portion of the ATP binding site on EF. EF is activated by the host calcium sensor, calmodulin. Surface plasmon resonance spectroscopic analysis shows that this compound does not affect the binding of calmodulin to EF. This compound is dissimilar from a previously described, non-nucleoside inhibitor of host adenylyl cyclase. It may serve as a lead to design antitoxins to address the role of adenylyl cyclase toxins in bacterial pathogenesis and to fight against anthrax and whooping cough.     

Ca(2+), Sr(2+), and Ba(2+) identify distinct regulatory sites on adenylyl cyclase (AC) types VI and VIII and consolidate the apposition of capacitative cation entry channels and Ca(2+)-sensitive ACs

Ca(2+)-sensitive adenylyl cyclases may act as early integrators of the two major second messenger-signaling pathways mediated by Ca(2+) and cAMP. Ca(2+) stimulation of adenylyl cyclase type I (ACI) and adenylyl cyclase type VIII (ACVIII) is mediated by calmodulin and the site on these adenylyl cyclases that interacts with calmodulin has been defined. By contrast, the mechanism whereby Ca(2+) inhibits adenylyl cyclase type V (ACV) and adenylyl cyclase type VI (ACVI) is unknown. In this study, Ca(2+), Sr(2+), and Ba(2+) were compared to probe the involvement of E-F hand-like domains in both Ca(2+) stimulation and inhibition of ACVIII and ACVI, respectively. HEK 293 cells transfected with ACVIII cDNA and C6-2B glioma cells (where the endogenous adenylyl cyclases is predominantly ACVI) were used to compare the effects of these three cations in in vitro and in vivo measurements. The in vitro data identified two Ca(2+) regulatory sites for both ACVIII and ACVI. Strikingly different potency series for these cations at mediating high affinity stimulation and inhibition of ACVIII and ACVI, respectively, effectively rule out the possibility that calmodulin or proteins utilizing similar Ca(2+)-binding motifs mediate inhibition of ACVI. On the other hand, the low affinity inhibition that is common to both ACVIII and ACVI showed virtually identical potency profiles for the IIa cation series, indicating a common site of action. Remarkably, whereas Sr(2+) was rather ineffective at regulating these cyclases (particularly ACVI) in vitro, adequate concentrations accumulated in the vicinity of these enzymes as a consequence of capacitative cation entry to partially regulate both of these activities in vivo. This latter finding consolidates earlier observations that Ca(2+)-sensitive adenylyl cyclases detect and respond to capacitative cation entry rather than global cytosolic cation concentrations.     

Tyrphostins are inhibitors of guanylyl and adenylyl cyclases

Guanylyl cyclase C (GC-C), the receptor for guanylin, uroguanylin, and the heat-stable enterotoxin, regulates fluid balance in the intestine and extraintestinal tissues. The receptor has an extracellular domain, a single transmembrane spanning domain, and an intracellular domain that harbors a region homologous to protein kinases, followed by the C-terminal guanylyl cyclase domain. Adenine nucleotides can regulate the guanylyl cyclase activity of GC-C by binding to the intracellular kinase homology domain (KHD). In this study, we have tested the effect of several protein kinase inhibitors on GC-C activity and find that the tyrphostins, known to be tyrosine kinase inhibitors, could inhibit GC-C activity in vitro. Tyrphostin A25 (AG82) was the most potent inhibitor with an IC(50) of approximately 15 microM. The mechanism of inhibition was found to be noncompetitive with respect to both the substrate MnGTP and the metal cofactor. Interestingly, the activity of the catalytic domain of GC-C (lacking the KHD) expressed in insect cells was also inhibited by tyrphostin A25 with an IC(50) of approximately 5 microM. As with the full-length receptor, inhibition was found to be noncompetitive with respect to MnGTP. Inhibition was reversible, ruling out a covalent modification of the receptor. Structurally similar proteins such as the soluble guanylyl cyclase and the adenylyl cyclases were also inhibited by tyrphostin A25. Evaluation of a number of tyrphostins allowed us to identify the requirement of two vicinal hydroxyl groups in the tyrphostin for effective inhibition of cyclase activity. Therefore, our studies are the first to report that nucleotide cyclases are inhibited by tyrphostins and suggest that novel inhibitors based on the tyrphostin scaffold can be developed, which could aid in a greater understanding of nucleotide cyclase structure and function.     

Guanylyl cyclases, a growing family of signal-transducing enzymes.

Guanylyl cyclases, which catalyze the formation of the intracellular signal molecule cyclic GMP from GTP, display structural features similar to other signal-transducing enzymes such as protein tyrosine-kinases and protein tyrosine-phosphatases. So far, three isoforms of mammalian membrane-bound guanylyl cyclases (GC-A, GC-B, GC-C), which are stimulated by either natriuretic peptides (GC-A, GC-B) or by the enterotoxin of Escherichia coli (GC-C), have been identified. These proteins belong to the group of receptor-linked enzymes, with different NH2-terminal extracellular receptor domains coupled to a common intracellular catalytic domain. In contrast to the membrane-bound enzymes, the heme-containing soluble guanylyl cyclase is stimulated by NO and NO-containing compounds and consists of two subunits (alpha 1 and beta 1). Both subunits contain the putative catalytic domain, which is conserved in the membrane-bound guanylyl cyclases and is found twice in adenylyl cyclases. Coexpression of the alpha 1- and beta 1-subunit is required to yield a catalytically active enzyme. Recently, another subunit of soluble guanylyl cyclase was identified and designated beta 2, revealing heterogeneity among the subunits of soluble guanylyl cyclase. Thus, different enzyme subunits may be expressed in a tissue-specific manner, leading to the assembly of various heterodimeric enzyme forms. The implications concerning the physiological regulation of soluble guanylyl cyclase are not known, but different mechanisms of soluble enzyme activation may be due to heterogeneity among the subunits of soluble guanylyl cyclase.     

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.