Synechocystis strain PCC 6803 cya2, a prokaryotic gene that encodes a guanylyl cyclase

Abstract

Synechocystis strain PCC 6803 exhibits similar levels of cyclic AMP (cAMP) and cyclic GMP (cGMP). A thorough analysis of its genome showed that Cya2 (Sll0646) has all the sequence determinants required in terms of activity and purine specificity for being a guanylyl cyclase. Insertional mutagenesis of cya2 caused a marked reduction in cGMP content without altering the cAMP content. Thus, Cya2 represents the first example of a prokaryotic guanylyl cyclase.

FIG. 1

(A) Domain architectures of class III purine nucleotide cyclases representative of eukaryotes (IC, ACG, rGC, sGC and sAC) and of their homologues in Synechocystis strain PCC 6803 (Cya1, Cya2, and Cya3 [PID1652963, PID1652044, and PID1652908, respectively]). Purine nucleotide cyclase catalytic domains are drawn as grey boxes. Red triangles indicate the reaction centres that contain all the potential determinants for catalysis (D, N, and R) and specificity (K for ACs and E for GCs). The colors of the amino acids refer to those that appear in the alignment shown in panel B. ACs are on the right, and GCs are on the left. The forkhead associated (FHA) and ferredoxin-like (Fd) domains are also indicated. The membrane is drawn in blue, and transmembrane helices are represented by orange barrels. A blue dot indicates the position of the N terminus. (B) Clustal W alignment of the AC/GC catalytic domains. The sequences correspond to a membrane mammalian AC (IC, PID117785) with its two domains (IC1 and IC2), the domain of a rat AC (IIC2, PID117786) whose three-dimensional structure has been determined, a homodimeric membrane AC from D. discoideum (ACG, PID399320), a soluble mammalian AC with its two domains (sAC1 and sAC2, PID4140400), a homodimeric membrane retinal GC (rGC), and the α and β subunits of a heterodimeric soluble GC (sGCα and sGCβ [PID118059 and PID118056, respectively]). Sequences are preceded by the percentage of identity with respect to IC2 and the position of the first residue in the corresponding sequence used for the alignment. When the two domains required for the building up of the catalytic centers are part of the same polypeptide chain, the sequence of the second domain is italicized. Boxes show the potential determinants for nucleotide specificity. The amino acids essential for catalysis are in green and double underlined. Numbering of the key residues refer to their position in the IC sequence, IC1 or IC2 depending of the domain that carries them. The residues discussed in the text are underlined. Numbers in parentheses correspond to residues not shown in the alignment. The topology and architecture of the eukaryotic proteins were obtained from GenBank. The FHA and AC/GC catalytic domains of the cyanobacterial polypeptides were detected using SMART. The Fd domain was identified using PSI-BLAST, and the prediction of transmembrane helices and topology of Cya2 were carried out using HMMTOP.

FIG. 2

Physical map of the region that surrounds cya2 (sll0646) on the Synechocystis strain PCC 6803 genome. The large arrows represent open reading frames, and the locations of the primers used for the PCR are indicated above the genes. The restriction sites used for the construction of the cya2 mutant are drawn. The omega cassette was inserted into the HincII site.

FIG. 3

Cyclic nucleotide levels in cultures of Synechocystis strain PCC 6803 and two independent cya2 mutants. Data (averages ± standard deviations) correspond to three independent cultures of each strain measured in triplicate.