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 hydrolase (EC 3.6.1.17) is a specific catabolic enzyme of Ap(4)A found in Schizosaccharomyces pombe. We have previously described the partial purification of Ap(4)A hydrolase from S. pombe [Robinson, de la Pena and Barnes (1993) Biochim. Biophys. Acta 1161, 139-148]. We determined the sequence of the N-terminal 20 amino acids of Ap(4)A hydrolase and designed two degenerate PCR primers based on the sequence. The 60 bp DNA fragment obtained by PCR, which is specific to Ap(4)A hydrolase, was used to isolate the Ap(4)A hydrolase gene, aph1, from S. pombe by screening a genomic DNA library in a multicopy plasmid. Ap(4)A hydrolase activity from the crude supernatant of a positive S. pombe transformant was about 25-fold higher than the control. There was no detectable stimulation of enzymic activity by phosphate. The aph1 gene from S. pombe contains three introns. The intron boundaries were confirmed by sequencing the cDNA of the aph1 gene from a S. pombe cDNA library. The deduced open reading frame of the aph1 gene codes for 182 amino acids. Two regions 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 prokaryotes, yeast, plants and mammals. Their functions are unknown, except that the bovine protein inhibits protein kinase C in vitro. All four histidine residues which are conserved among the HIT proteins, including the HxHxH putative Zn2+-binding motif, are conserved in the Ap(4)A hydrolase. In addition, there are two regions of similarity between the Ap(4)A phosphorylases I and II from Saccharomyces cerevisiae and Ap(4)A hydrolase from S. pombe. These regions overlap with the HIT protein similarity regions. The aph1 gene from S. pombe is the first asymmetrical Ap(4)A hydrolase gene to be cloned and sequenced.