CLONING OF THE SCHIZOSACCHAROMYCES-POMBE GENE ENCODING DIADENOSINE 5',5'''-P-1,P-4-TETRAPHOSPHATE (AP(4)A) ASYMMETRICAL HYDROLASE - SEQUENCE SIMILARITY WITH THE HISTIDINE TRIAD (HIT) PROTEIN FAMILY

Diadenosine 5',5'''-P-1,P-4-tetraphosphate (Ap(4)A) asymmetric hydrola se (EC 3.6.1.17) is a specific catabolic enzyme of Ap(4)A found in Sch izosaccharomyces pombe. We have previously described the partial purif ication of Ap(4)A hydrolase from S. pombe [Robinson, de la Pena and Ba rnes (1993) Biochim. Biophys. Acta 1161, 139-148]. We determined the s equence of the N-terminal 20 amino acids of Ap(4)A hydrolase and desig ned two degenerate PCR primers based on the sequence. The 60 bp DNA fr agment obtained by PCR, which is specific to Ap(4)A hydrolase, was use d to isolate the Ap(4)A hydrolase gene, aph1, from S. pombe by screeni ng a genomic DNA library in a multicopy plasmid. Ap(4)A hydrolase acti vity from the crude supernatant of a positive S. pombe transformant wa s about 25-fold higher than the control. There was no detectable stimu lation of enzymic activity by phosphate. The aph1 gene from S. pombe c ontains three introns. The intron boundaries were confirmed by sequenc ing the cDNA of the aph1 gene from a S. pombe cDNA library. The deduce d open reading frame of the aph1 gene codes for 182 amino acids. Two r egions of significant local similarity were identified between the Ap( 4)A hydrolase and the histidine triad (HIT) protein family [Seraphin ( 1992) DNA Sequence 3, 177-179]. HIT proteins are present in prokaryote s, yeast, plants and mammals. Their functions are unknown, except that the bovine protein inhibits protein kinase C in vitro. All four histi dine residues which are conserved among the HIT proteins, including th e HxHxH putative Zn2+-binding motif, are conserved in the Ap(4)A hydro lase. In addition, there are two regions of similarity between the Ap( 4)A phosphorylases I and II from Saccharomyces cerevisiae and Ap(4)A h ydrolase from S. pombe. These regions overlap with the HIT protein sim ilarity regions. The aph1 gene from S. pombe is the first asymmetrical Ap(4)A hydrolase gene to be cloned and sequenced.