D. Maksel et al., CLONING AND EXPRESSION OF DIADENOSINE 5',5'''-P-1,P-4-TETRAPHOSPHATE HYDROLASE FROM LUPINUS-ANGUSTIFOLIUS L, Biochemical journal, 329, 1998, pp. 313-319
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.