The Saccharomyces cerevisiae YOR163w Gene Encodes a Diadenosine 5′,5‴-P 1,P 6-Hexaphosphate (Ap6A) Hydrolase Member of the MutT Motif (Nudix Hydrolase) Family

Abstract

The YOR163w open reading frame on chromosome XV of the Saccharomyces cerevisiae genome encodes a member of the MutT motif (nudix hydrolase) family of enzymes of M r 21,443. By cloning and expressing this gene in Escherichia coli and S. cerevisiae, we have shown the product to be a (di)adenosine polyphosphate hydrolase with a previously undescribed substrate specificity. Diadenosine 5,5ٟ-P 1 ,P 6 -hexaphosphate is the preferred substrate, and hydrolysis in H 2 18 O shows that ADP and adenosine 5-tetraphosphate are produced by attack at P ␤ and AMP and adenosine 5-pentaphosphate are produced by attack at P ␣ with a K m of 56 M and k cat of 0.4 s ؊1 . Diadenosine 5,5ٟ-P 1 ,P 5 -pentaphosphate, adenosine 5-pentaphosphate, and adenosine 5-tetraphosphate are also substrates, but not diadenosine 5,5ٟ-P

Cloning, expression and characterization of YSA1H, a human adenosine 5′-diphosphosugar pyrophosphatase possessing a MutT motif

“…It therefore seems that recombinant YSA1H migrates anomalously on SDS\PAGE. This phenomenon of slower migration has been observed with other MutT motif proteins, including human Ap % A hydrolase [6] and S. cere isiae Ap ' A hydrolase [18] and might result from incomplete denaturation and SDS binding during sample preparation owing to the stability imparted by the mixed β-sheet core structure that might be a feature of this enzyme family [23]. (Figure 6b, lane 3).…”

“…Fluoride is a potent inhibitor of several nudix hydrolases involved in diadenosine polyphosphate metabolism [7,18,26]. Because it also inhibits YSA1H with an IC &!…”

“…N-terminal sequencing and electrospray mass spectrometric analysis were performed as described previously [6,18].…”

“…Substrates were screened by measuring the P i released in a coupled assay by co-incubation of substrate with YSA1H protein and either alkaline phosphatase or inorganic pyrophosphatase [16,18]. The standard assay (200 µl) for phosphodiester substrates was incubation for 10 min at 37 mC with 50 mM Tris\HCl (pH 7.5)\5 mM MgCl # \1 mM dithiothreitol\0.3 mM substrate\ 0.25 µg of Trx-YSA1H fusion protein and 0.5 µg (1 unit) of alkaline phosphatase.…”

The g5R (D250) Gene of African Swine Fever Virus Encodes a Nudix Hydrolase That Preferentially Degrades Diphosphoinositol Polyphosphates

“…They all possess the Nudix sequence signature motif Gx 5 Ex 5 [UA]xREx 2 EExGU (where U is an aliphatic hydrophobic amino acid), formerly known as the MutT motif (1,14). It is likely that these enzymes play the dual roles of removing potentially toxic nucleotides and nucleotide metabolites (e.g., oxidized nucleotides, ADP-ribose) from the cellular pools and of adjusting the levels of nucleotide cofactors and messengers in response to metabolic pressures and requirements (1).…”