Publications

• Expression and biochemical characterization of the human enzyme N-terminal asparagine amidohydrolase.

  Cantor J.R., Stone E.M., Georgiou G.

  The enzymatic deamidation of N-terminal L-Asn by N-terminal asparagine amidohydrolase (NTAN1) is a feature of the ubiquitin-dependent N-end rule pathway of protein degradation, which relates the in vivo half-life of a protein to the identity of its N-terminal residue. Herein, we report the bacteri ... >> More

  The enzymatic deamidation of N-terminal L-Asn by N-terminal asparagine amidohydrolase (NTAN1) is a feature of the ubiquitin-dependent N-end rule pathway of protein degradation, which relates the in vivo half-life of a protein to the identity of its N-terminal residue. Herein, we report the bacterial expression, purification, and biochemical characterization of human NTAN1 (hNTAN1). We show here that hNTAN1 is highly selective for the hydrolysis of N-terminal peptidyl L-Asn but fails to deamidate free L-Asn or L-Gln, N-terminal peptidyl L-Gln, or acetylated N-terminal peptidyl L-Asn. Similar to other N-terminal deamidases, hNTAN1 is shown to possess a critical Cys residue that is absolutely required for catalysis, corroborated in part by abolishment of activity through the Cys75Ala point mutation. We also present evidence that the exposure of a conserved L-Pro at the N-terminus of hNTAN1 following removal of the initiating L-Met is important for the function of the enzyme. The results presented here should assist in the elucidation of molecular mechanisms underlying the neurological defects of NTAN1-deficient mice observed in other studies, and in the discovery of potential physiological substrates targeted by the enzyme in the modulation of protein turnover via the N-end rule pathway. << Less

  Biochemistry 50:3025-3033(2011) [PubMed] [EuropePMC]

• A mouse amidase specific for N-terminal asparagine. The gene, the enzyme, and their function in the N-end rule pathway.

  Grigoryev S., Stewart A.E., Kwon Y.T., Arfin S.M., Bradshaw R.A., Jenkins N.A., Copeland N.G., Varshavsky A.

  The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. In both fungi and mammals, the tertiary destabilizing N-terminal residues asparagine and glutamine function through their conversion, by enzymatic deamidation, into the secondary destabilizing resi ... >> More

  The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. In both fungi and mammals, the tertiary destabilizing N-terminal residues asparagine and glutamine function through their conversion, by enzymatic deamidation, into the secondary destabilizing residues aspartate and glutamate, whose destabilizing activity requires their enzymatic conjugation to arginine, one of the primary destabilizing residues. We report the isolation and analysis of a mouse cDNA and the corresponding gene (termed Ntan1) that encode a 310-residue amidohydrolase (termed NtN-amidase) specific for N-terminal asparagine. The approximately 17-kilobase pair Ntan1 gene is located in the proximal region of mouse chromosome 16 and contains 10 exons ranging from 54 to 177 base pairs in length. The approximately 1.4-kilobase pair Ntan1 mRNA is expressed in all of the tested mouse tissues and cell lines and is down-regulated upon the conversion of myoblasts into myotubes. The Ntan1 promoter is located approximately 500 base pairs upstream of the Ntan1 start codon. The deduced amino acid sequence of mouse NtN-amidase is 88% identical to the sequence of its porcine counterpart, but bears no significant similarity to the sequence of the NTA1-encoded N-terminal amidohydrolase of the yeast Saccharomyces cerevisiae, which can deamidate either N-terminal asparagine or glutamine. The expression of mouse NtN-amidase in S. cerevisiae nta1Delta was used to verify that NtN-amidase retains its asparagine selectivity in vivo and can implement the asparagine-specific subset of the N-end rule. Further dissection of mouse Ntan1, including its null phenotype analysis, should illuminate the functions of the N-end rule, most of which are still unknown. << Less

  J. Biol. Chem. 271:28521-28532(1996) [PubMed] [EuropePMC]

• Protein NH2-terminal asparagine deamidase. Isolation and characterization of a new enzyme.

  Stewart A.E., Arfin S.M., Bradshaw R.A.

  An apparently unique enzyme, designated protein NH2-terminal asparagine deamidase (PNAD), that specifically converts NH2-terminal asparagine residues of peptide and protein substrates to aspartic acid, has been isolated to homogeneity from porcine liver by an eight-step procedure. PNAD is a relati ... >> More

  An apparently unique enzyme, designated protein NH2-terminal asparagine deamidase (PNAD), that specifically converts NH2-terminal asparagine residues of peptide and protein substrates to aspartic acid, has been isolated to homogeneity from porcine liver by an eight-step procedure. PNAD is a relatively low abundance protein, is readily solubilized, and exists as a monomeric species of approximately 33 kDa. PNAD does not act on internal asparagine residues and requires a free N alpha-amino group. It has reduced or no activity on NH2-terminal asparagine dipeptides and no activity toward free asparagine or asparagine amide. It does not act on any NH2-terminal glutamine substrates. PNAD does not show a strong pH dependence suggesting that the enzyme can act equally well on substrates with ionized or unionized alpha-amino groups. The properties and specificity of PNAD are consistent with those expected for the enzyme required for the ubiquitin-dependent turnover of intracellular proteins that initiate with Met-Asn-. Such proteins should be N alpha-acetylated on the retained initiator methionine and can subsequently be modified by the removal of N-acetyl methionine by acylaminoacid hydrolase. Conversion of the resulting NH2-terminal asparagine to aspartic acid by PNAD would render the protein susceptible to arginylation, polyubiquitinylation and degradation as specified by the N-end rule. << Less

  J. Biol. Chem. 269:23509-23517(1994) [PubMed] [EuropePMC]