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 Å resolution. CPDase hydrolyzes ADP-ribose 1'',2''-cyclic phosphate (Appr>p), a product of the tRNA splicing reaction, to the monoester ADP-ribose 1''-phosphate (Appr-1''p). The 181 amino acid protein shows a novel, bilobal arrangement of two αβ modules. Each lobe consists of two α-helices on the outer side of the molecule, framing a three- or four-stranded antiparallel β-sheet in the core of the protein. The active site is formed at the interface of the two β-sheets in a water-filled 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 flap-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.