In response to a plant SN-38 signal present in nodules, three receptor-like adenylate cyclases CyaD1, CyaD2 and CyaK synthesize the secondary messenger molecule 3′, 5′cAMP. 3′, 5′cAMP together with the Crp-like transcriptional activator Clr in turn promote transcription of the target gene smc02178, of unknown biochemical function . We have recently found that this cascade contributes to the autoregulation of the symbiotic interaction. Specifically, activation of the cAMP cascade in nodules inhibits, by a mechanism that remains to be elucidated, secondary infection by rhizospheric bacteria.
This control is lost in either a triple cyaD1cyaD2cyaK mutant, a clr or a smc02178 mutant resulting in a hyper-infection phenotype on plants–ie an abundance of EPZ015938 cell line abortive ITs on roots–as a consequence of a relaxed control of secondary infection . The concentration of the second messenger 3′, 5′cAMP in cells is controlled at the level of its synthesis by ACs and/or by its degradation Lazertinib cost to 5′AMP by phosphodiesterases (PDEs). PDEs are a superfamily of enzymes divided in three, non-homologous, main classes. All mammalian PDEs as well as several enzymes identified in Drosophila, Caenorhabditis and Saccharomyces cerevisiae belong to class I, whose conserved
carboxy-terminal catalytic domain contains two invariant motifs H(X)3H(X)25-35D/E . Class II PDEs are enzymes from Saccharomyces cerevisiae, Dictyostelium discoideum, Schizosaccharomyces pombe, C. albicans, and Vibrio fischeri. This class of enzymes shares the conserved motif HXHLDH. Class III PDEs belong to the superfamily of metallophosphoesterases . They share the conserved sequence motif D-(X)n-GD(X)n-GNH[E/D]-(X)n-H-(X)n-GHXH
as well as a βαβαβ secondary structure signature Benzatropine . Here we report on the characterization of a class III PDE from S. meliloti (SpdA, SMc02179) that we anticipated from the localization of the spdA gene at the cyaD1 locus to be involved in signal termination by turning-over the secondary messenger 3′, 5′cAMP. We have found that purified SpdA had actually no detectable activity against 3′, 5′cAMP and, instead, had high activity on the structural isomer 2′, 3′cAMP, which may occur in cells as a by-product of RNA degradation . We demonstrated that, contrary to 3′, 5′cAMP that promoted Clr binding to a cognate binding-site, 2′, 3′cAMP bound unproductively to Clr. Although SpdA biological function remains to be established, we present circumstantial evidence that SpdA may insulate 3′, 5′cAMP-mediated signaling from 2′, 3′-structural isomers. Results SpdA, a putative PDE Inspection of the cyaD1 locus (Figure 1A), that contains the clr gene as well as the clr–target gene smc02178, pointed to the smc02179 gene product as a potential PDE that we subsequently coined SpdA.