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

The crystal structure of the cyclic phosphodiesterase (CPDase) from Arabido psis thaliana, an enzyme involved in the tRNA splicing pathway, was determi ned at 2.5 Angstrom resolution. CPDase hydrolyzes ADP-ribose 1 " ,2 " -cycl ic phosphate (Appr>p), a product of the tRNA splicing reaction, to the mono ester ADP-ribose 1 " -phosphate (Appr-1 "p). The 181 amino acid protein sho ws a novel, bilobal arrangement of two alpha beta modules. Each lobe consis ts of two alpha -helices on the outer side of the molecule, framing a three - or four-stranded antiparallel beta -sheet in the core of the protein. The active site is formed at the interface of the two beta -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-exp osed surface loop (residues 100-115) is very likely to play a flap-like rol e, opening and closing the active site. Based on the crystal structure and on recent mutagenesis studies of a homologous CPDase from Saccharomyces cer evisiae, we propose an enzymatic mechanism that employs the nucleophilic at tack of a water molecule activated by one of the active site histidines.