title: Structural basis for dual specificity of yeast N-terminal amidase in the N-end rule pathway.
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 KMand kcatfor 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.
authors: Department of Life Sciences, Korea University, Seoul 02841, Korea.
authors: Min Kyung
authors: MK
authors: Kim
authors: Department of Life Sciences, Korea University, Seoul 02841, Korea.
authors: Sun Joo
authors: SJ
authors: Oh
authors: Department of Life Sciences, Korea University, Seoul 02841, Korea.
authors: Byung-Gil
authors: BG
authors: Lee
authors: Department of Life Sciences, Korea University, Seoul 02841, Korea hksong@korea.ac.kr.
authors: Hyun Kyu
authors: 0000-0001-5684-4059
authors: HK
authors: Song
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D005973:Glutamine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D059748:Proteolysis; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; D005973:Glutamine; D001216:Asparagine; D000581:Amidohydrolases; C585586:NTA1 protein, S cerevisiae
keywords: N-end rule; Nta1; dual specificity; nitrilase superfamily