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

Abstract

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.

Fig. 1

Soluble adenylyl cyclase (sAC) orthologues in different species. a Diagram of eukaryotic and Chloroflexusaurantiacus adenylyl cyclases (ACs) along with bacterial ACs. The relative similarities are “expect values” taken directly from a PSI-BLAST search of the human sAC protein vs the non-redundant GenBank database (http://www.ncbi.nlm.nih.gov/BLAST/). These values estimate the statistical significance of the match by specifying the number of matches expected to occur by chance. The values indicating greatest similarity are highlighted in red for single-domain bacterial ACs. Relative locations of the catalytic domains within the bacterial ACs are represented as shaded boxes and are aligned under the sAC catalytic domain with greater similarity. P indicates the conserved P-loop nucleotide-binding motif. The number of amino acid residues are also indicated. b Phylogenetic relationship between catalytic domains from a variety of ACs aligned by Clustal W (Higgins et al. 1994) is represented as an unrooted dendrogram constructed by a neighbor-joining plot (Perriere and Gouy 1996). Default values at DNA Data Bank of Japan (http://www.ddbj.nig.ac.jp/search/clustalw-j.html) were used with 1,000 bootstrap replications. The numbers indicate bootstrap values, using Escherichia coli CYA as the outgroup. A scale of branch length is shown in the lower right corner. ACs known to be activated by bicarbonate are in red, those known to be bicarbonate insensitive are in blue. Accession numbers for the proteins used: P. falciparum β, NP_704518; C. aurantiacus Chlo1066, ZP_00018085; C. aurantiacus Chlo1187, ZP_00018205; C. aurantiacus Chlo1431, ZP_00018442; Spirulina platensis CyaA, BAA22996; S. platensis CyaC, T17197; Stigmatella aurantiaca CyaA, CAA11549; S. aurantiaca CyaB1, T10905; Synecocystis sp. PCC6803 CyaA2, BAA16969; Anabaena spirulensis CyaA, P43524; A. spirulensis CyaB1, ZP_00018205; A. spirulensis CyaB2, BAA13999; A. spirulensis CyaC, BAA14000; Mycobacterium leprae AC, CAA19149; Sinorhizobium melioti AC, S60684; Mesorhizobium loti Cya3, BAB50205; Dictyostelium discoideum SgcA, AAL92097; A. gambiae, EAA10271; Rattus norvegicus sAC, AF081941; human sAC, NP_060887; Mus musculus sAC, NP_766617; rabbit sAC, AAO38673; Mycobacterium Rv1264, CAB00890; Mycobacterium Rv1319c, Q10632; human tmAC4, AAM94373; bovine tmAC7, CAA89894; Mus musculus tmAC9, CAA90570; Ciona intestinalis AC, GciWno1004_n06; E. coli CYA, CAA47280

Fig. 2

Characterization of Chloroflexus sAC activity. a Coomassie Blue-stained 10% SDS-PAGE demonstrating the purity of Chloroflexus sAC protein used in this study. bChloroflexus sAC activity measured in the presence of different cations. cChloroflexus sAC activity measured as a function of substrate ATP-Mn²⁺ in the presence of excess MnCl₂ (100 mM) for 10 min. The K_m value of 0.7 mM ATP-Mn²⁺ was determined using non-linear regression analysis. Graphs shown are representative of three independent experiments performed in triplicates

Fig. 3

Bicarbonate activation of Chlo1187. a Sequence alignment of a portion of the catalytic domain of Chlo1187 with the homologous region of other cyclases. Arrowhead indicates the conserved threonine residue suggested to be responsible for bicarbonate activation. bChloroflexus sAC activity was assayed in the presence of 10 mM ATP and 5 mM MnCl₂ and indicated concentrations of NaHCO₃. Values represent averages of triplicate determinations, with error bars indicating SD from the means