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

Abdelghany, Hend M.; Bailey, Scott; Blackburn, G. Michael; Rafferty, John B.; McLennan, Alexander G.

doi:10.1074/jbc.m211983200

Cited by 17 publications

(16 citation statements)

References 38 publications

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“…This result is in agreement with previously reported mutant experiments about other enzymes (35)(36)(37). The reaction mechanism of Nudix proteins was proposed from the study of tertiary structure (2,3,38) and NMR (39 -41).…”

Section: Resultssupporting

confidence: 82%

The Nudix Hydrolase Ndx1 from Thermus thermophilus HB8 Is a Diadenosine Hexaphosphate Hydrolase with a Novel Activity

Iwai

Kuramitsu

Masui

2004

Journal of Biological Chemistry

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The ndx1 gene, which encodes a Nudix protein, was cloned from the extremely thermophilic bacterium Thermus thermophilus HB8. This gene encodes a 126-amino acid protein that includes the characteristic Nudix motif conserved among Nudix proteins. Ndx1 was overexpressed in Escherichia coli and purified. Ndx1 was stable up to 95°C and at extreme pH. Size exclusion chromatography indicated that Ndx1 was monomeric in solution. Ndx1 specifically hydrolyzed (di)adenosine polyphosphates but not ATP or diadenosine triphosphate, and it always generated ATP as the product. Diadenosine hexaphosphate (Ap 6 A), the most preferred substrate, was hydrolyzed to produce two ATP molecules, which is a novel hydrolysis mode for Ap 6 A, with a Optimal activity for Ap 6 A was observed at around pH 8.0 and about 70°C. We found two important residues with pK a values of 6.1 and 9.6 in the free enzyme and pK a values of 7.9 and 10.0 in the substrate-enzyme complex. Kinetic studies of proteins with amino acid substitutions suggested that Glu-46 and Glu-50 were conserved residues in the Nudix motif and were involved in catalysis. Trp-26 was likely involved in enzyme-substrate interactions based on fluorescence measurements. Based on these results, the mechanism of substrate recognition and catalysis are discussed.

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Section: Resultssupporting

confidence: 82%

The Nudix Hydrolase Ndx1 from Thermus thermophilus HB8 Is a Diadenosine Hexaphosphate Hydrolase with a Novel Activity

Iwai

Kuramitsu

Masui

2004

Journal of Biological Chemistry

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“…However, less attention has been focused on residues involved in substrate binding. 25) The aromatic residues, which stack one substrate adenosine moiety between them, are highly conserved in structure-based amino acid sequence alignments of animal and plant Ap 4 A hydrolases, 17) suggesting that both residues have important effects on substrate binding. The importance of stacking residues for substrate binding in C. elegans has been previously confirmed by replacing Tyr121 by Ala, which led to an 8-fold increase in wild-type K m value.…”

Section: Discussionmentioning

confidence: 99%

“…The importance of stacking residues for substrate binding in C. elegans has been previously confirmed by replacing Tyr121 by Ala, which led to an 8-fold increase in wild-type K m value. 25) Only one aromatic residue (Tyr87) is conserved in the Plasmodium enzyme and the other stacking equivalent residue is smaller Pro133, which creates additional space in the Plasmodium adenosine binding site compared with the human enzyme. An increase in the size of the adenosine binding cavity of the Plasmodium enzyme (by replacing Pro133 by Ala) led to a 5-fold increase in K m value due to loose stacking of the adenosine ring.…”

Section: Discussionmentioning

confidence: 99%

Molecular Characterization and Mutational Analysis of Recombinant Diadenosine 5′,5″-P1,P4-Tetraphosphate Hydrolase from Plasmodium falciparum

Osman

Endo

Oh‐hashi

et al. 2012

Biological & Pharmaceutical Bulletin

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“…A recent detailed mutagenesis study (43) of the catalytic and binding residues has highlighted several important residues within the binding site in C. elegans. Mutations H31V (equivalent to His-42 in human) and K83M (equivalent to Lys-94 in human) had the largest effect on K m , with corresponding increases to 110 and 140 M from the observed value of 8.8 M in the wild type enzyme.…”

Section: Discussionmentioning

confidence: 99%

Structure and Substrate-binding Mechanism of Human Ap4A Hydrolase

Swarbrick

Buyya

Gunawardana

et al. 2005

Journal of Biological Chemistry

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Asymmetric diadenosine 5,5ٟ-P 1 ,P 4 -tetraphosphate (Ap 4 A) hydrolases play a major role in maintaining homeostasis by cleaving the metabolite diadenosine tetraphosphate (Ap 4 A) back into ATP and AMP. The NMR solution structures of the 17-kDa human asymmetric Ap 4 A hydrolase have been solved in both the presence and absence of the product ATP. The adenine moiety of the nucleotide predominantly binds in a ring stacking arrangement equivalent to that observed in the x-ray structure of the homologue from Caenorhabditis elegans. The binding site is, however, markedly divergent to that observed in the plant/pathogenic bacteria class of enzymes, opening avenues for the exploration of specific therapeutics. Binding of ATP induces substantial conformational and dynamic changes that were not observed in the C. elegans structure. In contrast to the C. elegans homologue, important side chains that play a major role in substrate binding do not have to reorient to accommodate the ligand. This may have important implications in the mechanism of substrate recognition in this class of enzymes.Nudix (nucleoside diphosphate linked to x) hydrolases are a superfamily of enzymes with over 300 identified members (1) that are required for maintaining physiological homeostasis by the metabolism of signaling molecules and potentially toxic substances. This superfamily is subdivided according to substrate specificity, including dinucleotide polyphosphatases (including Ap 4 A 1 hydrolases), ribo-or deoxyribonucleoside triphosphatases, and pyrophosphatases of nucleotide sugars, NADH, and ADP-ribose. All of the Nudix enzyme structures solved to date display the same "prototypical" scaffold comprising an ␣-␤-␣ sandwich fold in which one of the helices and the loop preceding it make up the catalytic site for metal-mediated hydrolysis of the substrate. This loop-helix motif contains the generalized "Nudix signature," GX 5 EX 7 REUXEEXGU, in which U is a bulky aliphatic residue usually Ile, Leu, or Val. Although the folds are similar, loop and helix insertions are commonly observed outside of the highly conserved catalytic region. As a consequence, the family displays a range of nucleotide-binding positions even for similar substrates (2). Furthermore, dimeric enzymes are also observed in which the substrate can be found between adjacent monomers. Perhaps the most striking example of this to date is in the x-ray structure of ADP-ribose pyrophosphatase (3), which requires dimerization through extensive domain swapping for substrate recognition and catalytic activity. Hence, throughout evolution, nature has preserved the scaffold of the reaction sub-site and facilitated diverse substrate recognition by employing residue or loop insertions and in some cases adopted contributions from each monomer in the dimeric state.Nudix Ap 4 A hydrolases (hereafter referred to as Ap 4 Aases) have been isolated from all kingdoms of life (1). Phylogenetic analysis (4) further reveals two distinct groups within these kingdoms. Plant and bacterial enzymes fall ...

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

Cited by 17 publications

References 38 publications

The Nudix Hydrolase Ndx1 from Thermus thermophilus HB8 Is a Diadenosine Hexaphosphate Hydrolase with a Novel Activity

The Nudix Hydrolase Ndx1 from Thermus thermophilus HB8 Is a Diadenosine Hexaphosphate Hydrolase with a Novel Activity

Molecular Characterization and Mutational Analysis of Recombinant Diadenosine 5′,5″-P<sup>1</sup>,P<sup>4</sup>-Tetraphosphate Hydrolase from <i>Plasmodium falciparum</i>

Structure and Substrate-binding Mechanism of Human Ap4A Hydrolase

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