Engineered Monomeric Human Histidine Triad Nucleotide-binding Protein 1 Hydrolyzes Fluorogenic Acyl-adenylate and Lysyl-tRNA Synthetase-generated Lysyl-adenylate

Abstract

Hint1 is a homodimeric protein and member of the ubiquitous HIT superfamily. Hint1 catalyzes the hydrolysis of purine phosphoramidates and lysyl-adenylate generated by lysyl-tRNA synthetase (LysRS). To determine the importance of homodimerization on the biological and catalytic activity of Hint1, the dimer interface of human Hint1 (hHint1) was destabilized by replacement of Val 97 of hHint1 with Asp, Glu, or Arg. The mutants were shown to exist as monomers in solution by a combination of size exclusion chromatograph, static light scattering, and chemically induced dimerization studies. Circular dichroism studies revealed little difference between the stability of the V97D, V97E, and wild-type hHint1. Relative to wild-type and the V97E mutant, however, significant perturbation of the V97D mutant structure was observed. hHint1 was shown to prefer 3-indolepropionic acyl-adenylate (AIPA) over tryptamine adenosine phosphoramidate monoester (TpAd). Wild-type hHint1 was found to be 277-and 1000-fold more efficient (k cat /K m values) than the V97E and V97D mutants, respectively. Adenylation of wildtype, V97D, and V97E hHint1 by human LysRS was shown to correlate with the mutant k cat /K m values using 3-indolepropionic acyl-adenylate as a substrate, but not tryptamine adenosine phosphoramidate monoester. Significant perturbations of the active site residues were not detected by molecular dynamics simulations of the hHint1s. Taken together, these results demonstrate that for hHint1; 1) the efficiency (k cat / K m ) of acylated AMP hydrolysis, but not maximal catalytic turnover (k cat ), is dependent on homodimerization and 2) the hydrolysis of lysyl-AMP generated by LysRS is not dependent on homodimerization if the monomer structure is similar to the wild-type structure.Histidine triad nucleotide-binding proteins (Hint) 2 are members of the histidine triad (HIT) protein superfamily of nucleotidyltransferases and hydrolyases (1). HIT proteins are named for the conserved nucleotide binding motif containing the sequence His-X-His-X-His-XX, in which X is a hydrophobic amino acid. The HIT proteins have been classified into three subfamilies according to their enzymatic function, sequence composition, and structural similarity; the Hint branch, the fragile histidine triad (Fhit) branch, and the galactose-1-phosphate uridyltransferase (GalT) branch (1).The Hint branch is the most ancient and can be found in Archaea, Bacteria, and Eukaryotae. Hints are homodimeric proteins that have been found to be purine phosphoramidases (2-5). Recently, we have demonstrated that lysyl-adenylate (lysyl-AMP) generated by bacterial and human lysyl-tRNA synthetases (LysRS) are also substrates for the respective bacterial and human Hints (6). Escherichia coli hinT knock-out mutants exhibited salt-dependent cell growth, whereas Hint1 knock-out mice have an increased susceptibility to 7, 12-dimethylbenz[a]anthracene (DMBA)-induced ovarian and mammary tumors by the carcinogen DMBA and increased incidence of spontaneous tumors (2,7,8). In addition,...

Probing the Impact of the echinT C-Terminal Domain on Structure and Catalysis

“…1 Given the proximity of the C-terminus to the active site of echinT and our observation that it can adopt multiple conformations (vide supra), we chose to investigate the role of the C-terminus in catalysis by comparing the catalytic efficiencies of the deletion mutants Δ117-119 and Δ114-119. In addition, from previous studies of engineered monomeric hHint1 25 and C-terminal chimeras of hHint1 and echinT, 1 we suspected that the C-terminus may play a role in the hydrolysis of lysyl-AMP generated by E. coli LysRS.…”

Mechanism of Activation of PSI-7851 and Its Diastereoisomer PSI-7977

“…Cloning, Expression, and Purification of Hint1 and Hint3-Human histidine triad nucleotide-binding proteins 1 and 3 (Hint1 and Hint3) were amplified from human fetal poly(A) ϩ RNA (Clontech) and ligated into pET17b containing a preinserted N-terminal FLAG (DYKDDDK) affinity-tagged Escherichia coli dihydrofolate reductase gene followed by a PreScission protease cleavable linker site (PS) (GE Healthcare) (15,16). Human fetal poly(A) ϩ RNA was reverse-transcribed using a Superscript III first strand synthesis kit (Invitrogen) with random hexamers.…”

Evidence that Human Histidine Triad Nucleotide Binding Protein 3 (Hint3) is a Distinct Branch of the Histidine Triad (HIT) Superfamily

“…4,7 This approach facilitates the isolation of Hints that are unable to be purified by AMP affinity chromatography by taking advantage of the binding of ecDHFR to a methotrexate (MTX) affinity column. Two potential thrombin recognition sites were found in hHint3 sequence so the thrombin cleavable linker in pPH70D plasmid was replaced with the PreScission™ protease cleavable linker (GLGGGGGLVPRGTLEVLFQ/ GPLE) where the recognition sequences were underlined with cleavage occurring between Gln and Gly.…”

“…The Michaelis-Menten constants k cat and K m were determined for hHint3-1 and -2 with indolepropinoic acyl-adenylate (AIPA) 7 and a series of phosphoramidate substrates 5 (Tables 1 and 2 and Supplementary Fig. 4).…”

“…In addition to their nucleoside phosphoramidase activity, [2][3][4][5] Hint1 isolated from human and Escherichia coli has recently been shown to be an efficient aminoacyl-adenylate and acyl-adenylate hydrolase. 6,7 The regulation of aminoacyl-tRNA synthetase (AARS)-mediated aminoacyl-adenylate formation as well as AARS transcription factor association has been proposed as a possible physiological role for Hints. [8][9][10][11] Hint1 knockout mice have been shown to exhibit normal embryonic development.…”