Structural Evidence That Brain Cyclic Nucleotide Phosphodiesterase Is a Member of the 2H Phosphodiesterase Superfamily

Abstract

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 ␤-sheet and two ␣-helices. The ␤-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.The abundance of the enzyme 2Ј,3Ј-cyclic nucleotide 3Ј-phosphodiesterase (CNP 1 ; EC 3.1.4.37) in the central nervous system of all mammals and some other vertebrates such as amphibians and birds has long been an enigma. This derives from the continuing failure to identify a physiological substrate for this enzyme. CNP has an apparent specificity for nucleoside 2Ј,3Ј-cyclic phosphate, which it cleaves to 2Ј-nucleotide end products, none of which (with the exception of NADP/NADPH) are found in metabolite pools. The last 4 decades of research have failed to attribute a function to this protein, although many possibilities have been considered (extensively reviewed in Refs. 1-3). More recently, RICH, a neuronally associated homolog of CNP, has been discovered in fish (4, 5), and the catalytic active site of CNP has been investigated (6).CNP and RICH share catalytic features with three other groups of enzymes: fungal/plant RNA ligases involved in tRNA splicing (7,8), bacterial and archaeal RNA ligases (9) that ligate tRNA half-molecules containing 2Ј,3Ј-cyclic phosphate and 5Ј-hydroxyl termini, and plant and yeast cyclic phosphodiesterases (CPDases) that hydrolyze ADP-ribose 1Љ,2Љ-cyclic phosphate to yield ADP-ribose 1Ј-phosphate (at least one of these latter enzymes also hydrolyzes nucleoside 2Ј,3Ј-cyclic phosphates) (10, 11). These enzymes are thought to play a role in the tRNA-splicing pathways. The x-ray structures of a CPDase from Arabidopsis thaliana (12-14) and, most recently, 2Ј-5Ј RNA ligase from Thermus thermophilus (15) have been determined.Members of this enzyme superfamily occur across a vast range of organisms ranging from bacteria to mammals. It has been suggested (16) that all four classes of enzymes originated from a common ancestor because they all have two similarly spaced histidine-containing tetrapeptides; their catalytic domains have a similar size of ϳ200 residues with similar pattern of predicted secondary structural ...

Mammalian 2′,3′ cyclic nucleotide phosphodiesterase (CNP) can function as a tRNA splicing enzyme in vivo

“…X-ray and NMR structures of the catalytic fragment (CF) highlighted a bilobar fold composed of two repeated a + b modules related by pseudotwofold symmetry (Kozlov et al 2003;Sakamoto et al 2005). The two HxT motifs are located at the active site.…”

Identification of the Low Density Lipoprotein (LDL) Receptor-related Protein-1 Interactome in Central Nervous System Myelin Suggests a Role in the Clearance of Necrotic Cell Debris

“…CNP is an enzyme highly expressed in oligodendrocytes, with functions in tubulin polymerization and oligodendrocyte process outgrowth (17). CNP is an abundant component of myelin, accounting for up to 4% of the total protein content (18). Although myelination is normal in CNP knock-out mice, they succumb to axonal loss and neurodegeneration due to impaired communication between cells (19).…”

Iso-coenzyme A

“…This enzyme was probably 2Ј,3Ј-cyclic 3Ј-phosphodiesterase, which selectively produces 2Ј-phosphate nucleosides from their 2Ј,3Ј-cyclic species, such as NADP ϩ generation from cyclic-NADP ϩ (31). Although this enzyme constitutes ϳ4% of all myelin protein, to date the actual in vivo substrate for this enzyme's esterase activity has not been established (32,58,59). These reports suggest that isoCoA could possibly be generated enzymatically in vivo from either dephospho-CoA or 2Ј,3Ј-cyclic CoA; however, no definitive evidence for this notion yet exists.…”