Two amino acid substitutions convert a guanylyl cyclase, RetGC-1, into an adenylyl cyclase.
Guanylyl cyclases (GCs) and adenylyl cyclases (ACs) have fundamental roles in a wide range of cellular processes. Whereas GCs use GTP as a substrate to form cGMP, ACs catalyze the analogous conversion of ATP to cAMP. Previously, a model based on the structure of adenylate cyclase was used to predict the structure of the nucleotide-binding pocket of a membrane guanylyl cyclase, RetGC-1. Based on this model, we replaced specific amino acids in the guanine-binding pocket of GC with their counterparts from AC. A change of two amino acids, E925K together with C995D, is sufficient to completely alter the nucleotide specificity from GTP to ATP. These experiments strongly validate the AC-derived RetGC-1 structural model and functionally confirm the role of these residues in nucleotide discrimination.
Tucker
C L
CL
Howard Hughes Medical Institute and Department of Biochemistry, Box 357370, University of Washington, Seattle, WA 98195, USA.
Hurley
J H
JH
Miller
T R
TR
Hurley
J B
JB
D000262:Adenylyl Cyclases / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D004252:DNA Mutational Analysis; D006162:Guanylate Cyclase / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000235:genetics
D008565:Membrane Proteins; D000262:Adenylyl Cyclases; D006162:Guanylate Cyclase