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.

Exchange of substrate and inhibitor specificities between adenylyl and guanylyl cyclases.

The active sites of guanylyl and adenylyl cyclases are closely related. The crystal structure of adenylyl cyclase and modeling studies suggest that specificity for ATP or GTP is dictated in part by a few amino acid residues, invariant in each family, that interact with the purine ring of the substrate. By exchanging these residues between guanylyl cyclase and adenylyl cyclase, we can completely change the nucleotide specificity of guanylyl cyclase and convert adenylyl cyclase into a nonselective purine nucleotide cyclase. The activities of these mutant enzymes remain fully responsive to their respective stimulators, sodium nitroprusside and Gsalpha. The specificity of nucleotide inhibitors of guanylyl and adenylyl cyclases that do not act competitively with respect to substrate are similarly altered, indicative of their action at the active sites of these enzymes.

Salt-resistant hypertension in mice lacking the guanylyl cyclase-A receptor for atrial natriuretic peptide.

Around half of all humans with essential hypertension are resistant to salt (blood pressure does not change by more than 5 mm Hg when salt intake is high), and although various inbred strains of rats display salt-insensitive elevated blood pressure, a gene defect to account for the phenotype has not been described. Atrial natriuretic peptide (ANP) is released from the heart in response to atrial stretch and is thought to mediate its natriuretic and vaso-relaxant effects through the guanylyl cyclase-A receptor (GC-A). Here we report that disruption of the GC-A gene results in chronic elevations of blood pressure in mice on a normal salt diet. Unexpectedly, the blood pressure remains elevated and unchanged in response to either minimal or high salt diets. Aldosterone and ANP concentrations are not affected by the genotype. Therefore, mutations in the GC-A gene could explain some salt-resistant forms of essential hypertension and, coupled with previous work, further suggest that the GC-A signaling pathway dominates at the level of peripheral resistance, where it can operate independently of ANP.

Rhizobium meliloti adenylate cyclase: probing of a NTP-binding site common to cyclases and cation transporters.

Alignments between amino acid sequences of eukaryotic adenylate (ACase) and guanylate (GCase) cyclases and the prokaryotic Rhizobium meliloti ACase revealed four conserved regions. Two were the target of site-directed mutagenesis. A positive charge at position 44 converted the enzyme to GCase, a negative charge at this position had no effect. A second modification indicated that residues 107 and 124 contribute to the nucleoside triphosphate binding pocket's conformation. This latter region was used to scan protein sequences data banks. A similar region was detected in the family of E1-E2 type ATPases. Topographical resemblance between these ATPases, eukaryotic ACases and several transporters suggest that they evolved from a common ancestor.

From adenylate cyclase to guanylate cyclase. Mutational analysis of a change in substrate specificity.

Adenylate and guanylate cyclases, having different but related substrates, are a paradigm for the study of substrate discrimination. A prokaryotic adenylate cyclase gene, phylogenetically related to eukaryotic counterparts, was screened for mutants remodelling the enzyme's specificity. In a first step, a mutant was selected displaying a significant level of guanylate cyclase activity. This was due to a point mutation destroying most of the adenylate cyclase activity. A second selection step restored most of the original activity. This resulted from an additional mutation in the same region, thus permitting the first identification of a functional domain in adenylate and guanylate cyclases.

Rhizobium meliloti adenylate cyclase is related to eucaryotic adenylate and guanylate cyclases.

A gene from Rhizobium meliloti coding for an adenylate cyclase was sequenced, and the deduced protein sequence was compared with those of other known adenylate cyclases. No similarity could be detected with the procaryotic counterparts. However, striking similarity was found with the catalytic region of Saccharomyces cerevisiae adenylate cyclase, the cytoplasmic domains of bovine adenylate cyclase, and two mammalian guanylate cyclases. The gene was fused to the enteric beta-galactosidase, and the chimeric protein was purified by affinity chromatography. This fusion protein was found to direct the synthesis of cyclic AMP in vitro. This activity was strongly inhibited by the presence of GTP, but no cyclic GMP synthesis could be detected in conditions permitting cyclic AMP synthesis.