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-type ATPase, and the C terminus is a guanylyl cyclase with the membrane topology of canonical mammalian adenylyl cyclases, yet with the cytosolic loops, C1 and C2, inverted compared with the mammalian order. We expressed in Escherichia coli the cytoplasmic domains of the protozoan guanylyl cyclase, independently and linked by a peptide, as soluble proteins. The His(6)-tagged proteins were enriched by affinity chromatography and analyzed by immunoblotting. Guanylyl cyclase activity was reconstituted upon mixing of the recombinant C1a- and C2-positioned domains and in a linked C1a-C2 construct. Adenylyl cyclase activity was minimal. The nucleotide substrate specificity was switched from GTP to ATP upon mutation of the substrate defining amino acids Glu(1681) and Ser(1748) in the C1-positioned domain to the adenylyl cyclase specific amino acids Lys and Asp. Using the C2 domains of mammalian adenylyl cyclases type II or IX and the C2-positioned domain from the Paramecium guanylyl cyclase we reconstituted a soluble, all C2 adenylyl cyclase. All enzymes containing protozoan domains were not affected by Galpha(s)/GTP or forskolin, and P site inhibitors were only slightly effective.
Linder
J U
JU
Fakultät für Chemie und Pharmazie, Universität Tübingen, Auf der Morgenstelle 8, D-72076 Tübingen, Germany.
Hoffmann
T
T
Kurz
U
U
Schultz
J E
JE
D000262:Adenylyl Cyclases / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D005576:Colforsin / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006162:Guanylate Cyclase / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010247:Paramecium / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D011993:Recombinant Fusion Proteins; D005576:Colforsin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000262:Adenylyl Cyclases; D006162:Guanylate Cyclase