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 Arabidopsis thaliana, an enzyme involved in the tRNA splicing pathway, was determined at 2.5 A 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 alphabeta modules. Each lobe consists 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-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.
Hofmann
A
A
Protein Structure Section, Macromolecular Crystallography Laboratory, Program in Structural Biology, NCI-Frederick, Frederick, MD 21702, USA. hofmanna@ncifcrf.gov
Zdanov
A
A
Genschik
P
P
Ruvinov
S
S
Filipowicz
W
W
Wlodawer
A
A
D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000246:Adenosine Diphosphate Ribose / Q000031:analogs & derivatives* / Q000378:metabolism*; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012326:RNA Splicing; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D018749:RNA, Plant; C401161:adenosine diphosphate-ribose 1''-2''cyclic phosphate; D000246:Adenosine Diphosphate Ribose; D012343:RNA, Transfer; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases