Structure and mechanism of activity of the cyclic phosphodiesterase o 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.