Structure and mechanism of activity of the cyclic phosphodiesterase of Appr>p, a product of the tRNA splicing reaction

Abstract

The crystal structure of the cyclic phosphodiesterase (CPDase) from Arabidopsis thaliana, an enzyme involved in the tRNA splicing pathway, was determined at 2.5 A Ê resolution. CPDase hydrolyzes ADPribose 1¢¢,2¢¢-cyclic phosphate (Appr>p), a product of the tRNA splicing reaction, to the monoester ADPribose 1¢¢-phosphate (Appr-1¢¢p). The 181 amino acid protein shows a novel, bilobal arrangement of two ab modules. Each lobe consists of two a-helices on the outer side of the molecule, framing a three-or fourstranded antiparallel b-sheet in the core of the protein. The active site is formed at the interface of the two b-sheets in a water-®lled cavity involving residues from two H-X-T/S-X motifs. This previously noticed motif participates in coordination of a sulfate ion. A solvent-exposed surface loop (residues 100±115) is very likely to play a¯ap-like role, opening and closing the active site. Based on the crystal structure and on recent mutagenesis studies of a homologous CPDase from Saccharomyces cerevisiae, we propose an enzymatic mechanism that employs the nucleophilic attack of a water molecule activated by one of the active site histidines. Keywords: ADP-ribose 1¢¢,2¢¢-cyclic phosphate/ Arabidopsis/2¢,3¢-cyclic nucleotide phosphodiesterase/ tRNA splicing/X-ray crystallography

Biophysical Characterization of Cyclic Nucleotide Phosphodiesterases

“…This hypothesis was supported by the crystal structure (11,12) and a mutagenesis study with CPDase (S. cerevisiae) (4). Based on the current results, it is absolutely clear that these four residues are essential for the catalytic reaction since variation in either one of the four positions leads to almost complete reduction of interaction enthalpy.…”

“…The cloning strategy for PDase Homolog/RNA ligase from E. coli was similar to the one used for the yeast CPDase, using E. coli DNA and appropriate PCR primers; this protein was also expressed from a construct with pET28. All three proteins carried C-terminal hexa-histidine tags and were purified by affinity chromatography using a Ni 2ϩ -nitrilotriacetic acid resin as described in (11).…”

“…We recently reported the crystal structures for CPDase (A. thaliana) in its oxidized (11), semi-reduced, and inhibitor-bound forms (12). The overall structure consists of two almost symmetrical lobes formed by the non-contiguous parts of the peptide chain, where each lobe consists of a three-or four-stranded antiparallel ␤-sheet that constitutes the inner core of the protein.…”

Probing the equatorial groove of the hookworm protein and vaccine candidate antigen, Na-ASP-2

“…In close spatial vicinity of the tandem histidine motif (His88 and His148) are two acidic residues (Glu99 and Glu125). Such pairings of acidic with histidine residues have previously been observed in enzymatic sites, which also includes phosphoesterases (Hofmann et al, 2000b).…”

“…In particular, protease activity has been suggested as a shared feature of PR-1 proteins based on structure-function inference from the cone snail protease Tex31 (Milne et al, 2003), but could not be demonstrated to date. We were intrigued by the structural similarity of the tandem histidine motif of Group 1 ASPs with enzymatic sites found in phosphodiesterases such as the Arabidopsis CPDase (Hofmann et al, 2000b), and thus investigated the possibility that Na-ASP-2 might possess phosphohydrolase activity.…”