Adenosine Monophosphoramidase Activity of Hint and Hnt1 Supports Function of Kin28, Ccl1, and Tfb3

Abstract

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...

Gene Expression of Metabolic Enzymes and a Protease Inhibitor in the Prefrontal Cortex Are Decreased in Schizophrenia

“…One possibility is the HINT enzymes have adenylated proteins as substrates, although the potential ligands of HINT and subsequent reactants remain unknown. A loss of HINT activity restricted growth rate in yeast (19,21,23,24). Thus, a 35% reduction in HINT1 enzyme mRNA in neurons in cortical layer VI (21% decrease in layers II-V) might be the result of abnormal cell transcription, although results in yeast leading to alterations in cell growth rate do not translate into alterations in the growth of neurons that are terminally differentiated cells.…”

“…However, no grossly observable deficiencies were noted in hint Ϫ/Ϫ mice, suggesting that other redundant genes can functionally compensate (20). HINT enzyme homologues hydrolyze adenosine-5Ј-monophosphoramidate (AMPNH 2 ) to AMP plus the leaving NH 2 group (19,21). HINT enzyme activity is a positive regulator of Kin28, the human homologue is CDK7 (21).…”

“…HINT enzyme homologues hydrolyze adenosine-5Ј-monophosphoramidate (AMPNH 2 ) to AMP plus the leaving NH 2 group (19,21). HINT enzyme activity is a positive regulator of Kin28, the human homologue is CDK7 (21). Human CDK7 interacts with the transcriptional machinery in the nucleus (22).…”

Hint, Fhit, and GalT: Function, Structure, Evolution, and Mechanism of Three Branches of the Histidine Triad Superfamily of Nucleotide Hydrolases and Transferases

“…Specific substrates for rabbit Hint and its yeast ortholog, Hnt1, emerged alongside physiological assays for the yeast HNT1 gene (17). While deletion of hnt1 was tolerated by haploid yeast cells on glucose media at all temperatures and deletion of hnt1 did not lead to a galactose-phenotype at 30 °C, hnt1 deletion produced cells that are galactose-at 38-39 °C.…”

“…Just as yeast Hnt1 enzyme activity was destroyed by the H116A mutation, so the hnt1-H116A allele was without function in supporting growth of yeast cells on galactose at 39 °C. Because the Hnt1-H116A protein is not unstable, it was concluded that hydrolysis of an Hnt1 substrate is required for growth of yeast cells at 39 °C (17).…”