Structural basis for dual specificity of yeast N-terminal amidase in the N-end rule pathway
- Authors
- Kim, Min Kyung; Oh, Sun Joo; Lee, Byung-Gil; Song, Hyun Kyu
- Issue Date
- 1-11월-2016
- Publisher
- NATL ACAD SCIENCES
- Keywords
- dual specificity; nitrilase superfamily; N-end rule; Nta1
- Citation
- PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, v.113, no.44, pp.12438 - 12443
- Indexed
- SCIE
SCOPUS
- Journal Title
- PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
- Volume
- 113
- Number
- 44
- Start Page
- 12438
- End Page
- 12443
- URI
- https://scholar.korea.ac.kr/handle/2021.sw.korea/86880
- DOI
- 10.1073/pnas.1612620113
- ISSN
- 0027-8424
- Abstract
- The first step of the hierarchically organized Arg/N-end rule pathway of protein degradation is deamidation of the N-terminal glutamine and asparagine residues of substrate proteins to glutamate and aspartate, respectively. These reactions are catalyzed by the N-terminal amidase (Nt-amidase) Nta1 in fungi such as Saccharomyces cerevisiae, and by the glutamine-specific Ntaq1 and asparagine-specific Ntan1 Nt-amidases in mammals. To investigate the dual specificity of yeast Nta1 (yNta1)and the importance of second-position residues in Asn/Gln-bearing N-terminal degradation signals (N-degrons), we determined crystal structures of yNta1 in the apo state and in complex with various N-degron peptides. Both an Asn-peptide and a Gln-peptide fit well into the hollow active site pocket of yNta1, with the catalytic triad located deeper inside the active site. Specific hydrogen bonds stabilize interactions between N-degron peptides and hydrophobic peripheral regions of the active site pocket. Key determinants for substrate recognition were identified and thereafter confirmed by using structure-based mutagenesis. We also measured affinities between yNta1 (wild-type and its mutants) and specific peptides, and determined K-M and k(cat) for peptides of each type. Together, these results elucidate, in structural and mechanistic detail, specific deamidation mechanisms in the first step of the N-end rule pathway.
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