title: 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 (alpha + beta) fold rather than an (alpha/beta) 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 redox-dependent 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.
authors.affiliation: Protein Structure Section, Macromolecular Crystallography Laboratory, National Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland 21702, USA. hofmanna@ncifcrf.gov
authors.forename: Andreas
authors.initials: A
authors.lastname: Hofmann
authors.forename: Sergey
authors.initials: S
authors.lastname: Tarasov
authors.forename: Melissa
authors.initials: M
authors.lastname: Grella
authors.forename: Sergei
authors.initials: S
authors.lastname: Ruvinov
authors.forename: Fahd
authors.initials: F
authors.lastname: Nasr
authors.forename: Witold
authors.initials: W
authors.lastname: Filipowicz
authors.forename: Alexander
authors.initials: A
authors.lastname: Wlodawer
mesh_terms: D017360:Arabidopsis / Q000201:enzymology; D002151:Calorimetry; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D005453:Fluorescence; D008969:Molecular Sequence Data; D009712:Nucleotides, Cyclic / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D014461:Ultracentrifugation; D014508:Urea / Q000494:pharmacology
chemical_list: D009712:Nucleotides, Cyclic; D014508:Urea; D010727:Phosphoric Diester Hydrolases