Human Fhit (fragile histidine triad) protein, encoded by the FHIT putative tumor suppressor gene, is a typical dinucleoside 5',5"'-P1,P3-triphosphate (Ap3A) hydrolase (EC 3.6.1.29) on the basis of its enzymatic properties we report here. Ap3A is the preferred substrate among ApnA (n = 3-6), and AMP is always one of the reaction products. Mn2+ and Mg2+ are equally stimulatory, while Zn2+ is inhibitory with Ap3A as the substrate. Values of the K(m) for Ap3A and Ap4A are 1.3 and 4.6 microM, respectively. Values of the specificity constant, kcat/K(m), for Ap3A and Ap4A are 2.0 x 10(6) and 6.7 x 10(3) s-1 M-1, respectively, for a glutathione S-transferase (GST)-Fhit fusion protein. Site-directed mutagenesis of FHIT demonstrated that all four conserved histidines are required for full activity, and the central histidine of the triad is absolutely essential for Ap3A hydrolase activity. This putative tumor suppressor is the first evidence for a connection between dinucleotide oligophosphate metabolism and tumorigenesis. Also, Fhit is the first HIT protein in which the histidine residues have been demonstrated by mutagenesis to be critical for function.

ABSTRACT

Histidine triad nucleotide-binding protein (HINT), a dimeric purine nucleotide-binding protein from rabbit heart, is a member of the HIT (histidine triad) superfamily which includes HINT homologs and FHIT homologs. Crystal structures of HINT-nucleotide complexes demonstrate that the most conserved residues in the superfamily mediate nucleotide binding and that the HIT motif forms part of the phosphate binding loop. Galactose-1-phosphate uridylyltransferase, whose deficiency causes galactosemia, contains tandem HINT domains with the same fold and mode of nucleotide binding as HINT despite no overall sequence similarity. Features of FHIT, a diadenosine polyphosphate hydrolase and candidate tumor suppressor, are predicted from HINT-nucleotide structures.Histidine triad (HIT) proteins are a superfamily of proteins named for a His-ϕ-His-ϕ-His-ϕ-ϕ motif near the C-terminus in which ϕ is a hydrophobic amino acid 1 . This motif was reported be a binding site for zinc ion 2 . Primary sequence alignment of HIT proteins ( Fig. 1) reveals that there are two branches. One branch contains representatives in all three domains of cellular life, the eukarya, bacteria, and archaea. The second branch appears to be confined to eukarya, with representative sequences in humans and two yeast genera.We purified HINT (histidine triad nucleotide-binding protein), a member of the first branch of the HIT superfamily, as a dimeric purine nucleoside and nucleotide-binding protein from rabbit heart cytosol (J.G., N. Liang & J.M.L, in prep.). HINT is nearly identical to proteins that have been given the designation protein kinase C inhibitor-1 (PKCI-1). Bovine PKCI-1 was so named because it was present in brain cytosol fractions that inhibited a mixture of PKC isoforms 3 . It has not been possible to reproduce this inhibition with HINT from rabbit heart or with purified recombinant HINT (J.G., N. Liang & J.M.L., in prep.). The corresponding human cDNA was cloned in two independent two-hybrid screens. In the first screen, interactions were sought with the product of ATDC 4 , a cDNA that when overexpressed, suppresses the radiation-sensitivity of a fibroblast cell line from a child with AtaxiaTelangiectasia. In the second screen, interactions were sought with the N-terminal 317 amino acid regulatory domain of PKC isozyme β 5 . These authors refer to the protein as PKCI-1 (protein kinase C interacting protein) on account of the yeast two-hybrid interaction. The human gene has been mapped to 5q31.2 6 .Correspondence should be addressed to C.B., (telephone 215-503-4573, facsimile 215-923-2117, email brenner@dada.jci.tju.edu We report here the crystal structures of HINT in its unliganded form, as well as HINT bound to adenosine, GMP and 8-Br-AMP. These structures show that the residues most conserved in the HIT superfamily are involved in binding nucleotides, and that the histidine triad, rather than constituting a zinc binding site 2, 5 , forms part of the phosphate-binding loop. Because HINT has no PKC inhibitory activity (J.G., N. Liang &...

The histidine triad superfamily of nucleotide hydrolases and nucleotide transferases consists of a branch of proteins related to Hint and Aprataxin, a branch of Fhitrelated 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 . Yeast strains constructed with specific loss of the Hnt1 active site fail to grow on galactose at elevated temperatures. Loss of Hnt1 enzyme activity also leads to hypersensitivity to mutations in Ccl1, Tfb3, and Kin28, which constitute the TFIIK kinase subcomplex of general transcription factor TFIIH and to mutations in Cak1, which phosphorylates Kin28. The target of Hnt1 regulation in this pathway was shown to be downstream of Cak1 and not to affect stability of Kin28 monomers. Functional complementation of all Hnt1 phenotypes was provided by rabbit Hint, which is only 22% identical to yeast Hnt1 but has very similar adenosine monophosphoramidase activity. Histidine triad (HIT)1 proteins are a superfamily of nucleotide-binding proteins named for a near C-terminal HXHXHXX motif (X is a hydrophobic amino acid) positioned at the ␣-phosphate of nucleotide substrates (1). The first branch of the superfamily is named for rabbit Hint, which had been purified as an abundant protein from cardiac cytosol by adenosine affinity chromatography (2) and shown to have homologs in all forms of life (1). Recently, Aprataxin, a gene located at 9p13 that is inactivated in ataxia with oculomotor apraxia, the second most common of the autosomal recessive ataxias, was identified as a member of the Hint branch of the HIT superfamily (3, 4). Human Fhit (5), which functions as a tumor suppressor protein in human (6 -9) and murine (10, 11) epithelial tissues, is the prototypical member of the second branch of the HIT superfamily. Fhit homologs have been found in fungi (12) and animals (13-16) and exhibit diadenosine polyphosphate hydrolase activity. A third branch of the HIT superfamily contains more distantly related nucleotide transferases including galactose-1-phosphate uridylyltransferase, which is the enzyme deficient in galactosemics (1), budding yeast diadenosine tetraphosphate phosphorylases ApaI and Apa2 (17), and adenylylsulfate:phosphate adenylyltransferase (18). Ironically, although Hint is the most ancient and widespread of the HIT proteins, reasonable Hint substrates remained unidentified, and the consequences of mutations in Hint or Hint orthologs were unknown (19).Recently, human Hint was identified as...

Common fragile sites form gaps at characteristic chromosome bands in metaphases from normal cells after aphidicolin induction. The distribution of common fragile sites parallels the positions of neoplasia-associated chromosomal rearrangements, prompting the proposal that fragility disposes to chromosomal rearrangements. Implicit in this hypothesis is that genes at fragile sites are altered by chromosome rearrangement and thus contribute to neoplastic growth. Chromosome band 3p14.2, encompassing the most inducible common fragile region, FRA3B, has been cloned and the FHIT gene, straddling FRA3B, characterized. The gene is inactivated by deletions in cancer-derived cell lines and primary tumors and Fhit protein is absent or reduced in lung, stomach, kidney, and cervical carcinomas, consistent with function as a tumor suppressor. FRA3B thus fulfills the prophecy that fragile site alterations contribute to the neoplastic process through inactivation of a tumor suppressor gene.