Hint, Fhit, and GalT: function, structure, evolution, and mechanism of three branches of the histidine triad superfamily of nucleotide hydrolases and transferases

Abstract

HIT (histidine triad) proteins, named for a motif related to the sequence HphiHphiHphiphi (phi, a hydrophobic amino acid), are a superfamily of nucleotide hydrolases and transferases, which act on the alpha-phosphate of ribonucleotides, and contain a approximately 30 kDa domain that is typically either a homodimer of approximately 15 kDa polypeptides with two active-sites or an internally, imperfectly repeated polypeptide that retains a single HIT active site. On the basis of sequence, substrate specificity, structure, evolution, and mechanism, HIT proteins can be classified into the Hint branch, which consists of adenosine 5'-monophosphoramide hydrolases, the Fhit branch, which consists of diadenosine polyphosphate hydrolases, and the GalT branch, which consists of specific nucleoside monophosphate transferases, including galactose-1-phosphate uridylyltransferase, diadenosine tetraphosphate phosphorylase, and adenylyl sulfate:phosphate adenylytransferase. At least one human representative of each branch is lost in human diseases. Aprataxin, a Hint branch hydrolase, is mutated in ataxia-oculomotor apraxia syndrome. Fhit is lost early in the development of many epithelially derived tumors. GalT is deficient in galactosemia. Additionally, ASW is an avian Hint family member that has evolved to have unusual gene expression properties and the complete loss of its nucleotide binding site. The potential roles of ASW and Hint in avian sexual development are discussed elsewhere. Here we review what is known about biological activities of HIT proteins, the structural and biochemical bases for their functions, and propose a new enzyme mechanism for Hint and Fhit that may account for the differences between HIT hydrolases and transferases.

Figure 1

Structural definition of HIT proteins. A) Stereo view of the GalT half-barrel (blue) superimposed on the Hint dimer (green and pink). The half-barrel consists of nine (GalT) or ten (Hint) anti-parallel β strands that enclose a pair of anti-parallel α helices. GalT possesses a distinct loop structure and a helix that connects the N-terminal portion (right) of the half-barrel with the C-terminal portion (left) of the half-barrel. The GalT monomer has a single Hint-homologous active-site in its N-terminal portion below the large loop where UMP is bound. UMP bound to GalT superimposes with GMP bound in the right hand Hint monomer. B) Highly conserved residues in Hint define a new mode of nucleotide-binding on top of the β-sheet. One of the two identical active sites is shown bound to GMP (yellow). The figure is based on reported X-ray structures (2, 7) and analysis (7).

Figure 2

Substrates and forward reaction schemes for prototypical members of the three branches of the HIT superfamily. Note that Hint and Fhit hydrolyze nucleotidylated enzyme intermediates while the GalT nucleotidylated intermediate, stable to water, transfers to a second substrate, namely a phosphorylated sugar.

Figure 3

Models for regulation of Kin28 function by Hnt1 enzyme activity. See text for details.

Figure 4

Proposed catalytic mechanism for HIT hydrolases. Hint and Fhit substrates are depicted as AMP-X and their leaving groups as XH. Upon adenylylation of the middle His of the HIT motif, the atom at position X (a phosphoramide N for Hint substrates and the α-β bridging O for Fhit substrates) receives a low barrier hydrogen bond from the C-terminal His of the HIT motif. Transfer of this proton is postulated to favor leaving over readenylylation. The C-terminal His is then proposed to accept a low barrier hydrogen bond from the incoming water molecule that hydrolyzes the adenylylated intermediate.