Cloning and expression of diadenosine 5′,5′"-P1,P4-tetraphosphate hydrolase from Lupinus angustifolius L

The first isolation, cloning and expression of cDNA encoding an asymmetric diadenosine 5',5'''P-1,P-4-tetraphosphate pyrophosphohydrolase (Ap(4)A hydrolase) from a higher plant is described. Ap(4)A hydrolase protein was purified from seeds of both Lupinus luteus and Lupinus angustifolius and partially sequenced. The Ap(4)A hydrolase cDNA was cloned from L. angustifolius cotyledonary polyadenylated RNA using reverse transcription and PCR with primers based on the amino acid sequence. The 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 which was inhibited at concentrations of F-as low as 3 mu M. These are properties characteristic of Ap(4)A hydrolase (asymmetrical) (EC 3.6.1.17). Comparison of the Ap,A hydrolase sequences derived from the four known 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 from Schizosaccharomyces pombe, was detected in the Ag(4)A hydrolase from lupin.