A guanylyl cyclase from Paramecium with 22 transmembrane spans - Expression of the catalytic domains and formation of chimeras with the catalytic domains of mammalian adenylyl cyclases

Paramecium has a 280-kDa guanylyl cyclase. The N terminus resembles a P-typ e ATPase, and the C terminus is a guanylyl cyclase with the membrane topolo gy of canonical mammalian adenylyl cyclases, yet with the cytosolic loops, C1 and C2, inverted compared with the mammalian order. We expressed in Esch erichia coli the cytoplasmic domains of the protozoan guanylyl cyclase, ind ependently and linked by a peptide, as soluble proteins. The His(6)-tagged proteins were enriched by affinity chromatography and analyzed by immunoblo tting. Guanylyl cyclase activity was reconstituted upon mixing of the recom binant C1a- and C2-positioned domains and in a linked C1a-C2 construct, Ade nylyl cyclase activity was minimal. The nucleotide substrate specificity wa s switched from GTP to ATP upon mutation of the substrate defining amino ac ids Glu(1681) and Ser(1748) in the C1-positioned domain to the adenylyl cyc lase specific amino acids Lys and Asp. Using the C2 domains of mammalian ad enylyl cyclases type II or IX and the C2-positioned domain from the Paramec ium guanylyl cyclase we reconstituted a soluble, all C2 adenylyl cyclase, A ll enzymes containing protozoan domains were not affected by G alpha(s)/GTP or forskolin, and P site inhibitors were only slightly effective.