Biophysical Characterization of Cyclic Nucleotide Phosphodiesterases

Abstract

We have compared selected biophysical properties of three phosphodiesterases, from Arabidopsis thaliana, Saccharomyces cerevisiae, and Escherichia coli. All of them belong to a recently identified family of cyclic nucleotide phosphodiesterases. Experiments elucidating folding stability, protein fluorescence, oligomerization behavior, and the effects of substrates were conducted, revealing differences between the plant and the yeast protein. According to CD spectroscopy, the latter protein exhibits an (␣ ؉ ␤) fold rather than an (␣/␤) fold as found with CPDase (A. thaliana). The redox-dependent structural reorganization recently found for the plant protein by X-ray crystallography could not be detected by CD spectroscopy due to its only marginal effect on the total percentage of helical content. However, in the present study a redoxdependent effect was also observed for the yeast CPDase. The enzymatic activity of wild type CPDase (A. thaliana) as well as of four mutants were characterized by isothermal titration calorimetry and the results prove the requirement of all four residues of the previously identified tandem signature motif for the catalytic function. Within the comparison of the three proteins in this study, the PDase Homolog/RNA ligase (E. coli) shares more similarities with the plant than with the yeast protein.

Vanadium and proteins: Uptake, transport, structure, activity and function

“…similarities between 2",3"-cyclic uridine vanadate(V) and the cyclic phosphorylated substrates of CPDase, the obtained structural characterization confirms the previous proposed mechanism where His119 acts as the nucleophilic residue involved in the attack to the substrate [307][309].…”

“…Following the oxidized native structure (PDB: 1FSI) and the respective mechanism of action [309], the inhibitor-bound semi-reduced CPDase structure was obtained at 2.4 Å resolution using 2",3"-cyclic uridine vanadate(V) as inhibitor (PDB: 1JH7) [307]. Both forms share a common overall structure despite some minor differences due to the absence of a Cys104-Cys110 dissulfide bridge in the semi-reduced protein.…”

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

“…These enzymes are thought to play a role in the tRNA-splicing pathways. The x-ray structures of a CPDase from Arabidopsis thaliana (12)(13)(14) and, most recently, 2Ј-5Ј RNA ligase from Thermus thermophilus (15) have been determined.…”

Characterization of a heat-stable enzyme possessing GTP-dependent RNA ligase activity from a hyperthermophilic archaeon, Pyrococcus furiosus

“…Thus, conformational change in the b 79 and b 8 regions is likely to be required for effective recognition of the tRNA halves. In the crystal structure of a complex of CPDase from Arabidopsis thaliana and 29,39-cyclic uridine vanadate, there are two nucleoside-binding sites: the abovementioned active site and the second binding site, which is located outside of the cleft and close to the b 79 and b 8 regions (Hofmann et al 2002). Figure 7 shows that the region corresponding to the second binding site is affected by the addition of r .…”

“…This motif has been reported to be strictly conserved among members of the 2H phosphoesterase superfamily (Kato et al 2003;Sakamoto et al 2005). Biophysical analysis of the three phosphodiesterases (from Arabidopsis thaliana, yeast, and E. coli) has shown that CPDase activity depends on the presence of HXTX motifs (Hofmann et al 2002).…”