The bacterial class IIIb adenylate cyclase CyaC-Sm of Sinorhizobium meliloti: Redox sensing by a heme B binding membrane anchor

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Pro- and eukaryotic adenylate cyclases: Membrane anchors with sensory function?Adenylate cyclases (AC) producing the second messenger cyclic AMP from ATP are present in all classes of life (eukarya, bacteria and archaea). Cyclic AMP has a broad significance in regulating cellular functions. The AC enzymes are grouped in six classes, and the best characterized ACs are members of class III which covers most eukaryotic and bacterial ACs. The ACs of this class show a large variation in domain composition, signal perception, regulation and overall function. The proposal aims at understanding the function of CyaC-Sm of Sinorhizobium meliloti, a class IIIb AC, and the role of its hexa-helical (6-TM) membrane anchor in redox sensing. A 6-TM membrane anchor is conserved in the eukaryotic (including mammalian) and some bacterial class III ACs. Bacterial ACs of this design have not been characterized in molecular detail. Understanding CyaC-Sm may have an impact on assessing the membrane anchor and its potential sensory role in eukaryotic class III ACs. Preparatory work suggests that CyaC-Sm contains a quinone reactive heme B in the membrane anchor. Respiration and quinones are supposed to oxidize the heme B and an iron-sulfur cluster in a ferredoxin-type domain and, as a result, to control dimerization and activity of the catalytic domain. The membrane anchor with the quinone/heme site and the near-by FeS cluster show a remarkable similarity to domains of the well-characterized respiratory fumarate reductase in structural and functional terms. The role of the components and of a linked oxidoreductase (SMc01816) in controlling AC activity will be charac-terized with purified CyaC-Sm, and by the use of wild-type and mutant CyaC-Sm expressed in E. coli. Overall, the study aims at characterizing function and molecular details of CyaC-Sm as a novel, actually the first, redox-controlled class III adenylate cyclase. The work might be of broader significance by assigning the hexa-helical TM domain of AC proteins a sensory role which is also discussed for membrane-delimited eukaryotic ACs. The physiological role of CyaC-Sm in S. meliloti is not analyzed in the proposal. However, there are first indications that CyaC-Sm is linked to aerobic respiration and to denitrification.