CLONING AND EXPRESSION OF DIADENOSINE 5',5'''-P-1,P-4-TETRAPHOSPHATE HYDROLASE FROM LUPINUS-ANGUSTIFOLIUS L

The first isolation, cloning and expression of cDNA encoding an asymme tric diadenosine 5',5'''P-1,P-4-tetraphosphate pyrophosphohydrolase (A p(4)A hydrolase) from a higher plant is described. Ap(4)A hydrolase pr otein was purified from seeds of both Lupinus luteus and Lupinus angus tifolius and partially sequenced. The Ap(4)A hydrolase cDNA was cloned from L. angustifolius cotyledonary polyadenylated RNA using reverse t ranscription and PCR with primers based on the amino acid sequence. Th e cDNA encoded a protein of 199 amino acids, molecular mass 22982 Da. When expressed in Escherichia coli fused to a maltose-binding protein, the enzyme catalysed asymmetric cleavage of Ap(4)A to AMP and ATP whi ch was inhibited at concentrations of F-as low as 3 mu M. These are pr operties characteristic of Ap(4)A hydrolase (asymmetrical) (EC 3.6.1.1 7). Comparison of the Ap,A hydrolase sequences derived from the four k nown cDNAs from pig, human, lupin and fission yeast showed that, like the mammalian hydrolase, the lupin enzyme possesses a Mut T motif but no other significant similarities. No sequence similarity to the human fragile histidine triad protein, as found in the Ap(4)A hydrolase fro m Schizosaccharomyces pombe, was detected in the Ag(4)A hydrolase from lupin.