Kinetic, dynamic, ligand binding properties, and structural models of a dual-substrate specific nudix hydrolase from Schizosaccharomyces pombe.

Schizosaccharomyces pombe Aps1 is a nudix hydrolase that catalyzes the hydrolysis of both diadenosine 5',5'''-P(1),P(n)-oligophosphates and diphosphoinositol polyphosphates in vitro. Nudix hydrolases act upon a wide variety of substrates, despite having a common 23 amino acid catalytic motif; hence, the residues responsible for substrate specificity are considered to reside outside the common catalytic nudix motif. The specific residues involved in binding each substrate of S.

Correlation of fragile histidine triad (Fhit) protein structural features with effector interactions and biological functions.

We have previously shown that Fhit tumor suppressor protein interacts with Hsp60 chaperone machinery and ferredoxin reductase (Fdxr) protein. Fhit-effector interactions are associated with a Fhit-dependent increase in Fdxr stability, followed by generation of reactive oxygen species and apoptosis induction under conditions of oxidative stress. To define Fhit structural features that affect interactions, downstream signaling, and biological outcomes, we used cancer cells expressing Fhit mutants with amino acid substitutions that alter enzymatic activity, enzyme substrate binding, or phosphorylation at tyrosine 114.

Phosphorylation of the human Fhit tumor suppressor on tyrosine 114 in Escherichia coli and unexpected steady state kinetics of the phosphorylated forms.

The human tumor suppressor Fhit is a homodimeric histidine triad (HIT) protein of 147 amino acids which has Ap(3)A hydrolase activity. We have recently discovered that Fhit is phosphorylated in vivo and is phosphorylated in vitro by Src kinase [Pekarsky, Y., Garrison, P.

Fhit is a physiological target of the protein kinase Src.

The FHIT gene is a tumor suppressor that is frequently inactivated by genomic alterations at chromosomal region 3p14.2. In the last few years, a considerable amount of data describing inactivation of FHIT in a variety of human malignancies and demonstrating the tumor suppressor potential of Fhit have been reported.

Paralogous murine Nudt10 and Nudt11 genes have differential expression patterns but encode identical proteins that are physiologically competent diphosphoinositol polyphosphate phosphohydrolases.

We previously described paralogous human genes [NUDT10 and NUDT11 [where NUDT is (nucleoside diphosphate attached moiety 'X')-type motif, also known as the 'nudix'-type motif]] encoding type 3 diphosphoinositol polyphosphate phosphohydrolases (DIPP3) [Hidaka, Caffrey, Hua, Zhang, Falck, Nickel, Carrel, Barnes and Shears (2002) J. Biol. Chem.

Disruption and overexpression of the Schizosaccharomyces pombe aps1 gene, and effects on growth rate, morphology and intracellular diadenosine 5',5"'-P1,P5-pentaphosphate and diphosphoinositol polyphosphate concentrations.

Schizosaccharomyces pombe Aps1 is an enzyme that degrades both diadenosine oligophosphates (Ap(n)A, n =5 or 6) and diphosphoinositol polyphosphates [diphosphoinositol pentakisphosphate (PP-InsP(5)) and bisdiphosphoinositol tetrakisphosphate ([PP](2)-InsP(4))] in vitro. The in vivo substrates of Aps1 are unknown. We report here the identification of Ap(5)A, PP-InsP(5), [PP](2)-InsP(4) and a novel diphosphoinositol polyphosphate ([PP](x)-InsP(x)) in S.

An adjacent pair of human NUDT genes on chromosome X are preferentially expressed in testis and encode two new isoforms of diphosphoinositol polyphosphate phosphohydrolase.

Combinatorial expression of the various isoforms of diphosphoinositol synthases and phosphohydrolases determines the rates of phosphorylation/dephosphorylation cycles that have been functionally linked to vesicle trafficking, stress responses, DNA repair, and apoptosis. We now describe two new 19-kDa diphosphoinositol polyphosphate phosphohydrolases (DIPPs), named types 3alpha and 3beta, which possess the canonical Nudix-type catalytic motif flanked on either side by short Gly-rich sequences. The two enzymes differ only in that Pro-89 in the alpha form is replaced by Arg-89 in the beta form, making the latter approximately 2-fold more active in vitro.

Adenosine monophosphoramidase activity of Hint and Hnt1 supports function of Kin28, Ccl1, and Tfb3.

The histidine triad superfamily of nucleotide hydrolases and nucleotide transferases consists of a branch of proteins related to Hint and Aprataxin, a branch of Fhit-related hydrolases, and a branch of galactose-1-phosphate uridylyltransferase (GalT)-related transferases. Although substrates of Fhit and GalT are known and consequences of mutations in Aprataxin, Fhit, and GalT are known, good substrates had not been reported for any member of the Hint branch, and mutational consequences were unknown for Hint orthologs, which are the most ancient and widespread proteins in the Hint branch and in the histidine triad superfamily. Here we show that rabbit and yeast Hint hydrolyze the natural product adenosine-5'-monophosphoramidate (AMPNH(2)) in an active-site-dependent manner at second order rates exceeding 1,000,000 m(-1) s(-1).