Structural evidence that brain cyclic nucleotide phosphodiesterase is a member of the 2H phosphodiesterase superfamily.
2',3'-Cyclic-nucleotide 3'-phosphodiesterase (CNP) is an enzyme abundantly present in the central nervous system of mammals and some vertebrates. In vitro, CNP specifically catalyzes the hydrolysis of 2',3'-cyclic nucleotides to produce 2'-nucleotides, but the physiologically relevant in vivo substrate remains obscure. Here, we report the medium resolution NMR structure of the catalytic domain of rat CNP with phosphate bound and describe its binding to CNP inhibitors. The structure has a bilobal arrangement of two modules, each consisting of a four-stranded beta-sheet and two alpha-helices. The beta-sheets form a large cavity containing a number of positively charged and aromatic residues. The structure is similar to those of the cyclic phosphodiesterase from Arabidopsis thaliana and the 2'-5' RNA ligase from Thermus thermophilus, placing CNP in the superfamily of 2H phosphodiesterases that contain two tetrapeptide HX(T/S)X motifs. NMR titrations of the CNP catalytic domain with inhibitors and kinetic studies of site-directed mutants reveal a protein conformational change that occurs upon binding.
Kozlov
Guennadi
G
Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada.
Lee
John
J
Elias
Demetra
D
Gravel
Michel
M
Gutierrez
Pablo
P
Ekiel
Irena
I
Braun
Peter E
PE
Gehring
Kalle
K
D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D010455:Peptides; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases; D010727:Phosphoric Diester Hydrolases