title: Cloning and expression of diadenosine 5',5'''-P1,P4-tetraphosphate hydrolase from Lupinus angustifolius L.
abstract: The first isolation, cloning and expression of cDNA encoding an asymmetric diadenosine 5',5'''P1,P4-tetraphosphate pyrophosphohydrolase (Ap4A hydrolase) from a higher plant is described. Ap4A hydrolase protein was purified from seeds of both Lupinus luteus and Lupinus angustifolius and partially sequenced. The Ap4A 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 22982Da. When expressed in Escherichia coli fused to a maltose-binding protein, the enzyme catalysed asymmetric cleavage of Ap4A to AMP and ATP which was inhibited at concentrations of F- as low as 3 microM. These are properties characteristic of Ap4A hydrolase (asymmetrical) (EC 3.6.1. 17). Comparison of the Ap4A 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 Ap4A hydrolase from Schizosaccharomyces pombe, was detected in the Ap4A hydrolase from lupin.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3052, Australia.
authors.forename: D
authors.initials: D
authors.lastname: Maksel
authors.forename: A
authors.initials: A
authors.lastname: Guranowski
authors.forename: S C
authors.initials: SC
authors.lastname: Ilgoutz
authors.forename: A
authors.initials: A
authors.lastname: Moir
authors.forename: M G
authors.initials: MG
authors.lastname: Blackburn
authors.forename: K R
authors.initials: KR
authors.lastname: Gayler
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010944:Plants / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid
chemical_list: D017766:Acid Anhydride Hydrolases; C020023:bis(5'-nucleosyl)tetraphosphatase (asymmetrical)