Characterization of active-site residues in diadenosine tetraphosphate hydrolase from Lupinus angustifolius

Abstract

Site-directed mutagenesis has been used to characterize the functions of key amino acid residues in the catalytic site of the 'nudix' hydrolase, (asymmetrical) diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) hydrolase (EC 3.6.1.17) from Lupinus angustifolius, the three-dimensional solution structure of which has recently been solved. Residues within the nudix motif, Gly-(Xaa)5-Glu-(Xaa)7-Arg-Glu-Uaa-Xaa-(Glu)2-Xaa-Gly (where Xaa represents unspecified amino acids and Uaa represents the bulky aliphatic amino acids Ile, Leu or Val) conserved in 'nudix enzymes', and residues important for catalysis from elsewhere in the molecule, were mutated and the expressed proteins characterized. The results reveal a high degree of functional conservation between lupin asymmetric Ap4A hydrolase and the 8-oxo-dGTP hydrolase from Escherichia coli. Charged residues in positions equivalent to those that ligate an enzyme-bound metal ion in the E. coli 8-oxo-dGTP hydrolase [Harris, Wu, Massiah and Mildvan (2000) Biochemistry 39, 1655-1674] were shown to contribute to catalysis to similar extents in the lupin enzyme. Mutations E55Q, E59Q and E125Q all reduced kcat markedly, whereas mutations R54Q, E58Q and E122Q had smaller effects. None of the mutations produced a substantial change in the Km)for Ap4A, but several extensively modified the pH-dependence and fluoride-sensitivities of the hydrolase. It was concluded that the precisely positioned glutamate residues Glu-55, Glu-59 and Glu-125 are conserved as functionally significant components of the hydrolytic mechanism in both of these members of the nudix family of hydrolases.

Analysis of the Catalytic and Binding Residues of the Diadenosine Tetraphosphate Pyrophosphohydrolase from Caenorhabditis elegans by Site-directed Mutagenesis

“…The contributions of the other residues to catalysis were also confirmed by site-directed mutagenesis: E53Q, E56Q, and E44Q led to 10 4.7 -, 25-, and 14-fold decreases in k cat , respectively (20), whereas K39Q and R52Q produced 8-fold and Ͼ10 3 -fold reductions, respectively (22). The principle of this catalytic mechanism appears to be well conserved among the Nudix hydrolases, including the lupin and Bartonella bacilliformis Ap 4 A hydrolases (15)(16)(17)23), human MTH1 (24), yeast Dcp2p (25), and human NUDT3 (DIPP1) (26).…”

“…On the basis of the 10 5 -fold reduction in k cat , we previously proposed that Glu 56 was most likely to be the catalytic base that deprotonates the attacking water molecule. However, the structural equivalents of Glu 52 in the lupin Ap 4 A hydrolase (Glu 55 ) and in the E. coli MutT protein (Glu 53 ) have been proposed as the deprotonating base (11,16,20). Glu 103 , although not in the Nudix motif, is positioned close to it in the three-dimensional structure and coordinates two of the four Mg 2ϩ ions located in the catalytic site (27).…”

“…Previous mutational studies with Nudix hydrolases have highlighted the importance of the Glu residues within the Nudix motif for catalysis ( Fig. 1) (16,20,25,26,31,32). Not surprisingly, therefore, the E56Q mutation was found to result in a 10 5 -fold reduction in k cat and virtual abolition of detectable enzyme activity, exactly as was found for the equivalent residue (Glu 59 ) in the lupin Ap 4 A hydrolase; in contrast, the K m was unaffected, indicating that the mutation has no A, the positions of residues in the amino acid sequence; conserved residues of the Nudix motif, residues which were mutated, and the resulting mutations are shown in bold.…”

“…Early studies of both animal and plant Ap 4 A Nudix hydrolases employing a combination of substrate analogues and labeling with heavy isotopes of oxygen revealed the mechanism of hydrolysis to involve in-line nucleophilic attack of a water molecule at the P 4 (P␣) phosphate with subsequent breakage of the P 4 -(O)P 3 bond (8,(11)(12)(13)(14). Recently, the catalytic residues of the lupin Ap 4 A hydrolase involved in this process were identified by a combination of structural analysis and site-directed mutagenesis (15)(16)(17). This study supported the catalytic mechanism previously described in detail for the prototypical Nudix hydrolase, the E. coli MutT 8-oxo-dGTPase.…”

Adenosine-5'-O-phosphorylated and adenosine-5'-O-phosphorothioylated polyols as strong inhibitors of (symmetrical) and (asymmetrical) dinucleoside tetraphosphatases

“…Homogeneous recombinant (asymmetrical) Ap 4 A hydrolases (EC 3.6.1.17) from human [24], narrow-leafed lupin (Lupinus angustifolius) [25] and Caenorhabditis elegans [26] were obtained as described. The lupin enzyme was kindly donated by Dr D. Maksel and Dr K. Gayler (University of Melbourne, Melbourne, Australia).…”