CLONING AND CHARACTERIZATION OF THE ARABIDOPSIS CYCLIC PHOSPHODIESTERASE WHICH HYDROLYZES ADP-RIBOSE 1'',2''-CYCLIC PHOSPHATE AND NUCLEOSIDE 2',3'-CYCLIC PHOSPHATES

In eukaryotic cells, pre-tRNAs spliced by a pathway that produces a 3' ,5'-phosphodiester, 2'-phosphomonoester linkage contain a 2'-phosphate group adjacent to the tRNA anticodon. This S'-phosphate is transferre d to NAD to give adenosine diphosphate (ADP)-ribose 1 '',2 ''-cyclic p hosphate (Appr>p), which is subsequently metabolized to ADP-ribose 1 ' '-phosphate (Appr-1 '' p). The latter reaction is catalyzed by a cycli c phosphodiesterase (CPDase), previously identified in yeast and wheat . In the work presented here, we describe cloning of the Arabidopsis c DNA encoding the 20-kDa CPDase that hydrolyzes Appr>p to Appr-1 '' p. Properties of the bacterially overexpressed and purified Arabidopsis e nzyme are similar to those of wheat CPDase. In addition to their trans formation of Appr>p, both enzymes hydrolyze nucleoside 2',3'-cyclic ph osphates to nucleoside S'-phosphates. For the Arabidopsis CPDase, the apparent K-m values for Appr>p, A>p, C>p, G>p, and U>p are 1.35, 1.34, 2.38, 16.86, and 17.67 mM, respectively. Southern analysis indicated that CPDase in Arabidopsis is encoded by a single copy gene that is ex pressed, at different levels, in all Arabidopsis organs that were anal yzed. Indirect immunofluorescence, performed with transfected protopla sts, showed that CPDase is localized in the cytoplasm. Based on substr ate specificity and products generated, the plant enzyme differs from other known cyclic phosphodiesterases. The Arabidopsis CPDase does not have recognizable structural similarity or motifs in common with prot eins deposited in public data bases.