Substrate selection by class III adenylyl cyclases and guanylyl cyclases

The second messengers cAMP and cGMP are of central importance in signal transduction pathways. To assure pathway specificity adenylyl and guanylyl cyclases are highly selective for their substrates, ATP and GTP, respectively. The universal class III cyclases are equipped with a variety of purine-binding modes, which have been identified by structure determination and mutagenesis. Most selection mechanisms rely on a pair of residues which form hydrogen bonds to N1 and the N(6)-amino or O(6)-keto group of adenine and guanine, respectively. Furthermore, selection is supported by hydrogen bonds involving the peptide backbone and by constraints imposed by hydrophobic side-chains.     

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.     

Bicarbonate stimulated adenylyl cyclases

Bicarbonate ion is fundamental to the biology of all living organisms. HCO(3)(-) is vital to such diverse physiological processes as carbon fixation, cellular homeostasis, sperm maturation, and nucleotide synthesis. A defined subset of adenylyl cyclases identified in eukaryotes and prokaryotes are directly activated by HCO(3)(-). As such, cAMP represents the first identified biological effector for fluctuations in intracellular inorganic carbon levels. The identification of a signal transduction pathway activated by HCO(3)(-) has far reaching implications for understanding how the cell responds to fluctuations in this essential anion.     

Plant purine nucleotide cyclases

Cyclic nucleotides (cAMP and cGMP) play an essential role in many important cellular processes in prokaryotic and eukaryotic organisms. They are produced by purine nucleotide cyclases: adenylyl and guanylyl cyclases. They are classified as one of two distinct forms: soluble and bound to membranes. Beside the differences in enzyme localization, the domain structure and regulation of enzymes activity are also diverse. However, all cyclases possess three groups of important residues: substrate specifying residue, metal binding residues and transition state stabilization residues. The natural occurrence of cyclic nucleotides in plants is now established. It was shown that in higher plants cNMPs act as a second messengers in a large number of (patho)physiological responses. However, it is only recently that the first plant enzymes with AC and GC activity of the unique structure have been identified and functionally characterized. In this study a systematic analysis of all the known prokaryotic, fungal and animal cyclases was done and direct evidences for the presence AC and GC in plant cells were shown.     

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.     

Prokaryotic squalene-hopene cyclases can be converted to citronellal cyclases by single amino acid exchange

Squalene-hopene cyclases (SHCs) are prokaryotic enzymes that catalyse the cyclisation of the linear precursor squalene to pentacyclic hopene. Recently, we discovered that a SHC cloned from Zymomonas mobilis (ZMO-1548 gene product) has the unique property to cyclise the monoterpenoid citronellal to isopulegol. In this study, we performed saturation mutagenesis of three amino acids of the catalytic centre of ZMO-1548 (F428, F486 and W555), which had been previously identified to interact with enzyme-bound substrate. Replacement of F428 by tyrosine increased hopene formation from squalene, but isopulegol-forming activity was strongly reduced or abolished in all muteins of position 428. W555 was essential for hopene formation; however, three muteins (W555Y, W428F or W555T) revealed enhanced cyclisation efficiency with citronellal. The residue at position 486 turned out to be the most important for isopulegol-forming activity. While the presence of phenylalanine or tyrosine favoured cyclisation activity with squalene, several small and/or hydrophobic residues such as cysteine, alanine or isoleucine and others reduced activity with squalene but greatly enhanced isopulegol formation from citronellal. Replacement of the conserved aromatic residue corresponding to F486 to cysteine in other SHCs cloned from Z. mobilis (ZMO-0872), Alicyclobacillus acidocaldarius (SHC _Aac ), Acetobacter pasteurianus (SHC _Apa ), Streptomyces coelicolor (SHC _Sco ) and Bradyrhizobium japonicum (SHC _Bja ) resulted in more or less strong isopulegol-forming activities from citronellal. In conclusion, many SHCs can be converted to citronellal cyclases by mutagenesis of the active centre thus broadening the applicability of this interesting class of biocatalyst.     

Identification of bacterial guanylate cyclases

The ability of bacteria to use cGMP as a second messenger has been controversial for decades. Recently, nucleotide cyclases from Rhodospirillum centenum, GcyA, and Xanthomonas campestris, GuaX, have been shown to possess guanylate cyclase activities. Enzymatic activities of these guanylate cyclases measured in vitro were low, which makes interpretation of the assays ambiguous. Protein sequence analysis at present is insufficient to distinguish between bacterial adenylate and guanylate cyclases, both of which belong to nucleotide cyclases of type III. We developed a simple method for discriminating between guanylate and adenylate cyclase activities in a physiologically relevant bacterial system. The method relies on the use of a mutant cAMP receptor protein, CRP_G, constructed here. While wild‐type CRP is activated exclusively by cAMP, CRP_G can be activated by either cAMP or cGMP. Using CRP‐ and CRP_G‐dependent lacZ expression in two E. coli strains, we verified that R. centenum GcyA and X. campestris GuaX have primarily guanylate cyclase activities. Among two other bacterial nucleotide cyclases tested, one, GuaA from Azospillrillum sp. B510, proved to have guanylate cyclase activity, while the other one, Bradyrhizobium japonicum CyaA, turned out to function as an adenylate cyclase. The results obtained with this reporter system were in excellent agreement with direct measurements of cyclic nucleotides secreted by E. coli expressing nucleotide cyclase genes. The simple genetic screen developed here is expected to facilitate identification of bacterial guanylate cyclases and engineering of guanylate cyclases with desired properties. Proteins 2015; 83:799–804. © 2015 Wiley Periodicals, Inc.     

Novel hopanoid cyclases from the environment

Hopanoids are ubiquitous isoprenoid lipids found in modern biota, in recent sediments and in low-maturity sedimentary rocks. Because these lipids primarily are derived from bacteria, they are used as proxies to help decipher geobiological communities. To date, much of the information about sources of hopanoids has come from surveys of culture collections, an approach that does not address the vast fraction of prokaryotic communities that remains uncharacterized. Here we investigated the phylogeny of hopanoid producers using culture-independent methods. We obtained 79 new sequences of squalene-hopene cyclase genes (sqhC) from marine and lacustrine bacterioplankton and analysed them along with all 31 sqhC fragments available from existing metagenomics libraries. The environmental sqhCs average only 60% translated amino acid identity to their closest relatives in public databases. The data imply that the sources of these important geologic biomarkers remain largely unknown. In particular, genes affiliated with known cyanobacterial sequences were not detected in the contemporary environments analysed here, yet the geologic record contains abundant hopanoids apparently of cyanobacterial origin. The data also suggest that hopanoid biosynthesis is uncommon: < 10% of bacterial species may be capable of producing hopanoids. A better understanding of the contemporary distribution of hopanoid biosynthesis may reveal fundamental insight about the function of these compounds, the organisms in which they are found, and the environmental signals preserved in the sedimentary record.     

Adenylyl cyclases in the digestive system

Adenylyl cyclases (ACs) are a group of widely distributed enzymes whose functions are very diverse. There are nine known transmembrane AC isoforms activated by Gαs. Each has its own pattern of expression in the digestive system and differential regulation of function by Ca^2 + and other intracellular signals. In addition to the transmembrane isoforms, one AC is soluble and exhibits distinct regulation. In this review, the basic structure, regulation and physiological roles of ACs in the digestive system are discussed.     

Inhibition of 2,3-oxidosqualene cyclases.

Monocyclic and tricyclic compounds possessing a nitrogen atom situated at a position corresponding to the carbenium ion of high energy intermediates or transition states involved during cyclization of 2,3-oxidosqualene to tetra- and pentacyclic triterpenes have been synthesized. These compounds were tested as inhibitors of 2,3-oxidosqualene cycloartenol, lanosterol-, and beta(alpha)-amyrin-cyclases in vitro and in vivo, and their affinity was compared to that of formerly synthesized 8-aza-bicyclic compounds [Taton et al. (1986) Biochem. Biophys. Res. Commun. 138, 764-770]. A monocyclic N-alkyl-hydroxypiperidine was shown to be the strongest inhibitor of the series upon cycloartenol-cyclase (I50 = 1 microM) from maize embryos but was much less effective on the beta(alpha)-amyrin-cyclases from Rubus fruticosus suspension cultures or pea cotyledons. In contrast, 13-aza-tricyclic derivatives displayed little inhibition on 2,3-oxidosqualene cycloartenol-, lanosterol-, and beta(alpha)-amyrin-cyclases. The obtained data exemplify the differences existing in the cyclization process between cycloartenol- (lanosterol-) cyclases on one hand and beta(alpha)-amyrin-cyclases on the other. The results are discussed with respect to current mechanisms postulated for 2,3-oxidosqualene cyclization. Because of its activity in vivo and in vitro the monocyclic N-alkyl-hydroxypiperidine appears to be a potent and promising tool to study sterol biosynthesis regulation.     

Soluble guanylate cyclases in the retina

Soluble guanylate cyclase catalyzes the formation of cyclic GMP using GTP as substrate. It is now well established that soluble guanylate cyclase is highly activated by nitric oxide, and that many of the effects of nitric oxide on various cells and tissues are mediated through increased production of cyclic GMP. This review discusses the evidence for the presence of soluble guanylate cyclases in different classes of cells in vertebrate retina and the role of these enzymes in retinal physiology. It is concluded that the enzyme is present in nearly every class of cells in the retina and that it may be involved in signal transmission between some cells and in the modulation of signal transmission between others.     

Dimerization of mammalian adenylate cyclases.

Mammalian adenylate cyclases are predicted to possess complex topologies, comprising two cassettes of six transmembrane-spanning motifs followed by a cytosolic, catalytic ATP-binding domain. Recent studies have begun to provide insights on the tertiary assembly of these proteins; crystallographic analysis has revealed that the two cytosolic domains dimerize to form a catalytic core, while more recent biochemical and cell biological analysis shows that the two transmembrane cassettes also associate to facilitate the functional assembly and trafficking of the enzyme. The older literature had suggested that adenylate cyclases might form higher order aggregates, although the methods used did not necessarily provide convincing evidence of biologically relevant events. In the present study, we have pursued this question by a variety of approaches, including rescue or suppression of function by variously modified molecules, coimmunoprecipitation and fluorescence resonance energy transfer (FRET) analysis between molecules in living cells. The results strongly suggest that adenylate cyclases dimerize (or oligomerize) via their hydrophobic domains. It is speculated that this divalent property may allow adenylate cyclases to participate in multimeric signaling assemblies.     

Structural homology between virulence-associated bacterial adenylate cyclases

The primary structure of the calmodulin-sensitive adenylate cyclase toxin from Bacillus anthracis has been determined from the corresponding nucleotide sequence and compared to that of the homologous toxin secreted by Bordetella pertussis. The cya gene of Bacillus anthracis encodes an 800 amino acid (aa) protein beginning with an N-terminal signal peptide. The central part of the B. anthracis adenylate cyclase includes a region of striking homology with the N-terminal part of the B. pertussis enzyme. In this region a particularly well conserved 24-aa peptide and two other less homologous peptides have been identified. These data corroborate the immunological relatedness of the two enzymes and suggest that the two prokaryotic calmodulin-sensitive adenylate cyclases originate from a common ancestor.     

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.     

Divergent evolution of oxidosqualene cyclases in plants

Triterpenes are one of the largest classes of plant metabolites and have important functions. A diverse array of triterpenoid skeletons are synthesized via the isoprenoid pathway by enzymatic cyclization of 2,3-oxidosqualene. The genomes of the lower plants Chlamydomonas reinhardtii and moss (Physcomitrella patens) contain just one oxidosqualene cyclase (OSC) gene (for sterol biosynthesis), whereas the genomes of higher plants contain nine to 16 OSC genes. Here we carry out functional analysis of rice OSCs and rigorous phylogenetic analysis of 96 OSCs from higher plants, including Arabidopsis thaliana, Oryza sativa, Sorghum bicolor and Brachypodium distachyon. The functional analysis identified an amino acid sequence for isoarborinol synthase (OsIAS) (encoded by Os11g35710/OsOSC11) in rice. Our phylogenetic analysis suggests that expansion of OSC members in higher plants has occurred mainly through tandem duplication followed by positive selection and diversifying evolution, and consolidated the previous suggestion that dicot triterpene synthases have been derived from an ancestral lanosterol synthase instead of directly from their cycloartenol synthases. The phylogenetic trees are consistent with the reaction mechanisms of the protosteryl and dammarenyl cations which parent a wide variety of triterpene skeletal types, allowing us to predict the functions of the uncharacterized OSCs.