Engineered monomeric human histidine triad nucleotide-binding protein 1 hydrolyzes fluorogenic acyl-adenylate and lysyl-tRNA synthetase-generated lysyl-adenylate

Tsui Fen Chou, Ilya B. Tikh, Bruno A.C. Horta, Brahma Ghosh, Ricardo B. De Alencastro, Carston R Wagner

Research output: Contribution to journalArticle

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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 Val97 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 (kcat/Km 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 kcat/Km 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 (kcat/Km) of acylated AMP hydrolysis, but not maximal catalytic turnover (kcat), 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.

Original languageEnglish (US)
Pages (from-to)15137-15147
Number of pages11
JournalJournal of Biological Chemistry
Volume282
Issue number20
DOIs
StatePublished - May 18 2007

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Lysine-tRNA Ligase
Hydrolysis
Adenosine Monophosphate
Adenosine
Monomers
Dimerization
Bioactivity
Dimers
Light scattering
Molecular dynamics
Catalyst activity
Computer simulation
Substrates
phosphoramidic acid
lysyladenylate
human HINT1 protein
Proteins
Molecular Dynamics Simulation
Viperidae
Circular Dichroism

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Engineered monomeric human histidine triad nucleotide-binding protein 1 hydrolyzes fluorogenic acyl-adenylate and lysyl-tRNA synthetase-generated lysyl-adenylate. / Chou, Tsui Fen; Tikh, Ilya B.; Horta, Bruno A.C.; Ghosh, Brahma; De Alencastro, Ricardo B.; Wagner, Carston R.

In: Journal of Biological Chemistry, Vol. 282, No. 20, 18.05.2007, p. 15137-15147.

Research output: Contribution to journalArticle

Chou, Tsui Fen ; Tikh, Ilya B. ; Horta, Bruno A.C. ; Ghosh, Brahma ; De Alencastro, Ricardo B. ; Wagner, Carston R. / Engineered monomeric human histidine triad nucleotide-binding protein 1 hydrolyzes fluorogenic acyl-adenylate and lysyl-tRNA synthetase-generated lysyl-adenylate. In: Journal of Biological Chemistry. 2007 ; Vol. 282, No. 20. pp. 15137-15147.
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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 Val97 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 (kcat/Km 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 kcat/Km 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 (kcat/Km) of acylated AMP hydrolysis, but not maximal catalytic turnover (kcat), 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.",
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T1 - Engineered monomeric human histidine triad nucleotide-binding protein 1 hydrolyzes fluorogenic acyl-adenylate and lysyl-tRNA synthetase-generated lysyl-adenylate

AU - Chou, Tsui Fen

AU - Tikh, Ilya B.

AU - Horta, Bruno A.C.

AU - Ghosh, Brahma

AU - De Alencastro, Ricardo B.

AU - Wagner, Carston R

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AB - 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 Val97 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 (kcat/Km 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 kcat/Km 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 (kcat/Km) of acylated AMP hydrolysis, but not maximal catalytic turnover (kcat), 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.

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