CyaC, a redox‐regulated adenylate cyclase of Sinorhizobium meliloti with a quinone responsive diheme‐B membrane anchor domain

Abstract

Summary The nucleotide cyclase CyaC of Sinorhizobium meliloti is a member of class III adenylate cyclases (AC), a diverse group present in all forms of life. CyaC is membrane‐integral by a hexahelical membrane domain (6TM) with the basic topology of mammalian ACs. The 6TM domain of CyaC contains a tetra‐histidine signature that is universally present in the membrane anchors of bacterial diheme‐B succinate‐quinone oxidoreductases. Heterologous expression of cyaC imparted activity for cAMP formation from ATP to Escherichia coli, whereas guanylate cyclase activity was not detectable. Detergent solubilized and purified CyaC was a diheme‐B protein and carried a binuclear iron‐sulfur cluster. Single point mutations in the signature histidine residues caused loss of heme‐B in the membrane and loss of AC activity. Heme‐B of purified CyaC could be oxidized or reduced by ubiquinone analogs (Q0 or Q0H2). The activity of CyaC in bacterial membranes responded to oxidation or reduction by Q0 and O2, or NADH and Q0H2 respectively. We conclude that CyaC‐like membrane anchors of bacterial ACs can serve as the input site for chemical stimuli which are translated by the AC into an intracellular second messenger response.

A New Electron Shuttling Pathway Mediated by Lipophilic Phenoxazine via the Interaction with Periplasmic and Inner Membrane Proteins of Shewanella oneidensis MR-1

“…Since resazurin/resorufin is cell membrane permeable and the presence of resazurin resulted in the downregulation of fumarate reduction, nitrogen reduction, and sulfate reduction genes, resazurin likely entered the periplasm space and interacted with periplasm and inner membrane proteins. As a consequence, resazurin/resorufin may have altered the redox state of inner membrane quinones, and then, the cyclic AMP (cAMP) receptor protein and adenylate cyclase (CyaC) , could respond to the redox change of quinones, finally resulting in the downregulation of the above-mentioned genes. , …”

“…As a consequence, resazurin/resorufin may have altered the redox state of inner membrane quinones, and then, the cyclic AMP (cAMP) receptor protein 58 and adenylate cyclase (CyaC) 59,60 could respond to the redox change of quinones, finally resulting in the downregulation of the above-mentioned genes. 61,62 In addition, it was previously reported that Mtr gene expression was enhanced in the presence of soluble EMs, such as quinone and flavins. 29 These soluble EMs mainly reacted with outer-membrane proteins (OmcA and MtrC), and consequently, the high demand for electron donors due to the enhanced EET resulted in the upregulation of Mtr genes.…”

Interaction of adenylate cyclase CyaC with regulators Smc01817 and Clr ofSinorhizobium meliloti: cAMP as an ON/OFF trigger in sensor-regulator complexes?

“…The proteins were cloned in plasmids pUT18, pUT18C, pKT25 and pKNT25 for producing the T18 and T25 fragment for C- and N-terminal fusion, respectively (Klein et al 2023). For the BATCH assays, the bacteria were grown anaerobically in LB broth with 20 mM DMSO which produces highest activities (Wissig et al 2019) in microtiter plates to an OD578 0.6 to 0.9. The β-galactosidase assays (Monzel et al 2023; Wörner et al 2016) were performed as described (Klein et al 2023) and are presented as the mean (with standard deviation) from at least two biological and four technical replicates each.…”

The evolutionary conservation of eukaryotic membrane-bound adenylyl cyclase isoforms

“…A different picture emerges regarding associated domains which are not required for activity. In bacterial ACs numerous diverse N-terminal domains have been identified, among them membrane anchors of two, four or six predicted α-helices, and several distinct domains located between membrane anchor and catalytic domain ( Linder and Schultz, 2003 ; Schultz and Natarajan, 2013 ; Beltz et al, 2016 ; Bassler et al, 2018 ; Wissig et al, 2019 ). Thus, each of these bacterial AC isoforms probably is endowed with unique molecular features, which confer peculiar regulatory modalities, almost completely unexplored at this time (for representative samples see Figure 1 and ( Bassler et al, 2018 )).…”

“…Another interesting question is why many bacteria contain multiple class III AC isozymes, e.g., 16 in Mycobacterium tuberculosis or 28 in Sinorhizobium meliloti . Notably, the AC CyaC from Sinorhizobium is regulated by its hexahelical membrane domain which contains a di-heme-B entity integrated in its membrane domain enabling regulation by oxidation-reduction processes ( Wissig et al, 2019 ). The regulation of other bacterial ACs with hexahelical membrane domains is unknown.…”