N-terminal-acetylases (NATs) catalyse the covalent attachment of an acetyl moiety from acetyl-CoA to the free alpha-amino group at the N-terminus of a protein. This irreversible modification neutralizes the positive charge at the N-terminus, makes the N-terminal residue larger and more hydrophobic, and prevents its removal by hydrolysis. It may also play a role in membrane targeting and gene silencing. NatE is found in all eukaryotic organisms and plays an important role in chromosome resolution and segregation. It specifically targets N-terminal L-methionine residues attached to Lys, Val, Ala, Tyr, Phe, Leu, Ser, and Thr. There is some substrate overlap with EC 2.3.1.256, N-terminal methionine Nalpha-acetyltransferase NatC. In addition, the acetylation of Met followed by small residues such as Ser, Thr, Ala, or Val suggests a kinetic competition between NatE and EC 3.4.11.18, methionyl aminopeptidase. The enzyme also has the activity of EC 2.3.1.48, histone acetyltransferase, and autoacetylates several of its own lysine residues.
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
acetyl-CoA + an N-terminal-L-methionyl-L-leucyl-[protein] = an N-terminal-Nalpha-acetyl-L-methionyl-L-leucyl-[protein] + CoA
hNaa50p utilizes an ordered bi bi reaction of the Theorell-Chance type, substarte binding study and dynamics using NMR spectroscopy. Acetyl-CoA induces a conformational change that is required for the peptide to bind to the active site. Addition of peptide in the absence of acetyl-CoA does not alter the structure of the protein
N-terminal-acetylases (NATs) catalyse the covalent attachment of an acetyl moiety from acetyl-CoA to the free alpha-amino group at the N-terminus of a protein. This irreversible modification neutralizes the positive charge at the N-terminus, makes the N-terminal residue larger and more hydrophobic, and prevents its removal by hydrolysis. It may also play a role in membrane targeting and gene silencing. NatE is found in all eukaryotic organisms and plays an important role in chromosome resolution and segregation. It specifically targets N-terminal L-methionine residues attached to Lys, Val, Ala, Tyr, Phe, Leu, Ser, and Thr. There is some substrate overlap with EC 2.3.1.256, N-terminal methionine Nalpha-acetyltransferase NatC. In addition, the acetylation of Met followed by small residues such as Ser, Thr, Ala, or Val suggests a kinetic competition between NatE and EC 3.4.11.18, methionyl aminopeptidase. The enzyme also has the activity of EC 2.3.1.48, histone acetyltransferase, and autoacetylates several of its own lysine residues.
preferably the enzyme acetylates oligopeptides with N-termini Met-Leu-Xxx-Pro. Furthermore, the enzyme autoacetylates lysines 34, 37, and 140 in vitro as well as histone 4
ectopically expressed hNaa50 results, predominantly, in the N-terminal-acetylation of N-terminal Met (iMet) starting N-termini, including iMet-Lys, iMet-Val, iMet-Ala, iMet-Tyr, iMet-Phe, iMet-Leu, iMet-Ser, and iMet-Thr N-termini. Presence of a kinetic competition between Naa50 and Met-aminopeptidases
product inhibition, competitive versus acetyl-CoA, noncompetitive against peptide substrate well in line with a ternary complex mechanism. The non-competitive inhibition pattern observed when CoA is tested against peptide as variable substrate and at fixed concentrations of acetyl-CoA also rules out a ping-pong mechanism
UniProt ID Q9NX55, a protein with intrinsic NAA10 catalytic subunit inhibitory activity. HYPK and hNAA50 can bind to hNatA simultaneously to form a tetrameric hNatE/HYPK complex. hNatE displays an about 8.6fold decrease of Km, and an about 1.1fold decrease of kcat, with an overall 7.7fold increase of catalytic efficiency, compared to hNAA50. In the presence HYPK, hNatE displays an about 1.3fold decrease in Km, and an about 3.8fold decrease in kcat, with an overall 2.9fold decrease in catalytic efficiency compared to hNatE alone. The hNatE/HYPK structure reveals a negative cooperative mechanism. Over the HYPK and hNatA interaction interface within the tetrameric complex, polar interactions between HYPK-Glu74 and hNAA15-Tyr158, between the backbone carbonyl of HYPK-Thr100 and hNAA15-Lys687, between the backbone carbonyl of HYPK-Asn129 and hNAA15-Arg697, and between HYPK-Asn129 and hNAA15-Lys696 are observed
NAA50 and HYPK each contribute to NAA10 activity inhibition through structural alteration of the NAA10 substrate-binding site. NAA50 activity is increased through NAA15 tethering, but is inhibited by HYPK through structural alteration of the NatE substrate-binding site. hNAA50 and HYPK inhibit hNatA activity, and HYPK is dominant. The hNatE structure reveals molecular basis for enzyme crosstalk
NAA50 and HYPK each contribute to NAA10 activity inhibition through structural alteration of the NAA10 substrate-binding site. NAA50 activity is increased through NAA15 tethering, but is inhibited by HYPK through structural alteration of the NatE substrate-binding site. hNAA50 and HYPK inhibit hNatA activity, and HYPK is dominant. The hNatE structure reveals molecular basis for enzyme crosstalk
the acetyltransferase activity of San stabilizes the mitotic cohesin at the centromeres in a shugoshin-independent manner. The enzyme is specifically required for the maintenance of the centromeric cohesion in mitosis
the crystal structure of yeast NatA/Naa50 is used as a scaffold to uncover evolutionarily conserved catalytic crosstalk within the orthologous complexes in yeast and human, overview. NatA/Naa50 forms a stable complex through evolutionarily conserved interactions, yeast Naa50 alone is defective in activity due to compromised substrate binding. The Saccharomyces cerevisiae ScNaa15 auxiliary subunit of NatA displays a high degree of structure conservation with Schizosaccharomyces pombe SpNaa15 and human hNaa15. NatA-Naa50 from yeast and human make conserved interactions
there are seven known NAT types (NatA through NatG), each composed of one or more specific subunits and having specific substrates defined by the very first amino acid residue (serine, alanine, etc.)
yeast Naa50 alone is defective in activity due to compromised substrate binding. Evolutionarily conserved Naa15 TY mutants can disrupt NatA-Naa50 association
enzyme complex NatE co-translationally acetylates the N-terminus of half the proteome to mediate diverse biological processes, including protein half-life, localization, and interaction. The complex hNatE, comprising subunits Naa10 and Naa15 (NatA) and Naa50, is more active than hNAA50 alone
N-terminal acetylation (NTA) is among the most widespread co-translational modifications found in eukaryotic proteins. NTA is carried out by N-terminal acetyltransferases (NATs), which catalyze the transfer of an acetyl moiety from acetyl coenzyme A to the N-terminal amino group of the nascent polypeptides as they emerge from the ribosome. NTA is estimated to affect up to 90% of human proteins and influences their folding, localization, complex formation, and degradation, along with a variety of cellular functions ranging from apoptosis to gene regulation. NTA is an irreversible protein modification
NatA (EC 2.3.1.255) co-translationally acetylates the N-termini of over 40% of eukaryotic proteins and can associate with another catalytic subunit, Naa50, to form a ternary NatA/Naa50 dual enzyme complex (also called NatE). NatA/Naa50 forms a stable complex through evolutionarily conserved interactions, yeast Naa50 alone is defective in activity due to compromised substrate binding, mechanism, overview
the human N-terminal acetyltransferase E (NatE) contains NAA10 and NAA50 as catalytic subunits, and NAA15 auxiliary as subunit and associates with HYPK, a protein with intrinsic NAA10 inhibitory activity. hNatE and inhibitor HYPK form a tetrameric complex. Analysis of the molecular basis for how NatE and HYPK cooperate, cryo-EM structures of human NatE and NatE/HYPK complexes, overview. NAA50 and HYPK exhibit negative cooperative binding to NAA15 in vitro and in human cells by inducing NAA15 shifts in opposing directions. HYPK and hNAA50 can bind to hNatA simultaneously to form a tetrameric hNatE/HYPK complex
the human N-terminal acetyltransferase E (NatE) contains NAA10 and NAA50 as catalytic subunits, and NAA15 auxiliary as subunit and associates with HYPK, a protein with intrinsic NAA10 inhibitory activity. hNatE and inhibitor HYPK form a tetrameric complex. Analysis of the molecular basis for how NatE and HYPK cooperate, cryo-EM structures of human NatE and NatE/HYPK complexes, overview. NAA50 and HYPK exhibit negative cooperative binding to NAA15 in vitro and in human cells by inducing NAA15 shifts in opposing directions. HYPK and hNAA50 can bind to hNatA simultaneously to form a tetrameric hNatE/HYPK complex
the NatA/Naa50 complex contains two catalytic subunits and one auxiliary subunit for co-translational N-terminal acetylation, structure and mechanism of acetylation by the N-terminal dual enzyme NatA/Naa50 complex, overview. NatA-Naa50 interactions promote catalytic crosstalk between Naa10 and Naa50
the NatA/Naa50 complex contains two catalytic subunits and one auxiliary subunit for co-translational N-terminal acetylation, structure and mechanism of acetylation by the N-terminal dual enzyme NatA/Naa50 complex, overview. NatA-Naa50 interactions promote catalytic crosstalk between Naa10 and Naa50
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
enzyme bound to a native substrate peptide fragment (MLGPEGGRWG) and CoA, hanging drop vapor diffusion method, using 16% (w/v) PEG 8000, 20% (v/v) glycerol, and 40 mM potassium phosphate (monobasic, pH 5.0)
Naa50p bound to a native substrate peptide fragment and CoA, hanging drop vapor diffusion method, mixing of 12 mg/ml protein in 25 mM HEPES, pH 7.5, 100 mM NaCl, and 10mM dithiothreitol, with peptide MLGPEGGRWG solution, and crystallization solution, containing 16% PEG 8000, 20% glycerol, and 40 mM potassium phosphate, pH 5.0, in a 1:3:3 molar ratio, 20°C, 1-3 days, X-ray diffraction structure determination and analysis at 2.75 A resolution
recombinant GST-tagged full-length Naa50p from Escherichia coli strain BL21(DE3) by glutathione affinity chromatography and cleavage of the GST-tag by TEV protease, followed by ion exchange chromatography and gel filtration
recombinant expression of hNaa15 mutants in HeLa cells, recombinant expression of N-terminally His-tagged NatA in Spodoptera frugiperda Sf9 cells, recombinant expression of His-tagged hNaa50 in Escherichia coli strain Rosetta (DE3)pLysS and strain BL21(DE3), coexpression of tagged HYPK
Naa50p is a therapeutic anti-cancer target, the structure of the ternary Naa50p complex also provides a molecular scaffold for the design of NAT-specific small molecule inhibitors with possible therapeutic applications
N-terminal acetylome analysis reveals the specificity of Naa50 (Nat5) and suggests a kinetic competition between N-terminal acetyltransferases and methionine aminopeptidases
Human Naa50 protein displays broad substrate specificity for amino-terminal acetylation: Detailed structural and biochemical analysis using tetrapeptide library
From molecular understanding to organismal biology of N-terminal acetyltransferases
Structure
27
1053-1055
2019
Homo sapiens (Q9GZZ1 AND P41227 AND Q9BXJ9), Saccharomyces cerevisiae (Q08689 AND P41227 AND P12945), Saccharomyces cerevisiae, Saccharomyces cerevisiae ATCC 204508 (Q08689 AND P41227 AND P12945), Schizosaccharomyces pombe, Schizosaccharomyces pombe 972, Schizosaccharomyces pombe ATCC 24843
Structure and mechanism of acetylation by the N-terminal dual enzyme NatA/Naa50 complex
Structure
27
1057-1070.e4
2019
Homo sapiens (Q9GZZ1 AND P41227 AND Q9BXJ9), Homo sapiens, Saccharomyces cerevisiae (Q08689 AND P07347 AND P12945), Saccharomyces cerevisiae, Saccharomyces cerevisiae ATCC 204508 (Q08689 AND P07347 AND P12945), Schizosaccharomyces pombe, Schizosaccharomyces pombe 972, Schizosaccharomyces pombe ATCC 24843