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Literature summary for 2.3.1.258 extracted from

  • Liszczak, G.; Arnesen, T.; Marmorsteins, R.
    Structure of a ternary Naa50p (NAT5/SAN) N-terminal acetyltransferase complex reveals the molecular basis for substrate-specific acetylation (2011), J. Biol. Chem., 286, 37002-37010.
    View publication on PubMedView publication on EuropePMC

Application

Application Comment Organism
medicine 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 Homo sapiens

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli BL21(DE3) cells Homo sapiens
expression of GST-tagged full-length Naa50p with a TEV protease-cleavable site in Escherichia coli strain BL21(DE3) Homo sapiens

Crystallization (Commentary)

Crystallization (Comment) Organism
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) Homo sapiens
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 Homo sapiens

Protein Variants

Protein Variants Comment Organism
F27A site-directed mutagenesis, inactive mutant Homo sapiens
F27A the mutant shows less than 10% of wild type catalytic efficiency Homo sapiens
F35A site-directed mutagenesis, inactive mutant Homo sapiens
F35A the mutant shows less than 10% of wild type catalytic efficiency Homo sapiens
H112A site-directed mutagenesis, inactive mutant Homo sapiens
H112A the mutant shows less than 10% of wild type catalytic efficiency Homo sapiens
H112F site-directed mutagenesis, inactive mutant Homo sapiens
H112F the mutant shows less than 10% of wild type catalytic efficiency Homo sapiens
I142A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Homo sapiens
I142A the mutant shows 42.2% of wild type catalytic efficiency Homo sapiens
P28A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Homo sapiens
P28A the mutant shows less 13.4% of wild type catalytic efficiency Homo sapiens
V29A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Homo sapiens
V29A the mutant shows 12.3% of wild type catalytic efficiency Homo sapiens
Y139A site-directed mutagenesis, inactive mutant Homo sapiens
Y139A the mutant shows less than 10% of wild type catalytic efficiency Homo sapiens
Y31A site-directed mutagenesis, inactive mutant Homo sapiens
Y31A the mutant shows less than 10% of wild type catalytic efficiency Homo sapiens
Y73A site-directed mutagenesis, inactive mutant Homo sapiens
Y73A the mutant shows less than 10% of wild type catalytic efficiency Homo sapiens
Y73F site-directed mutagenesis, inactive mutant Homo sapiens
Y73F the mutant shows less than 10% of wild type catalytic efficiency Homo sapiens

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
acetyl-CoA + MLGPEGGRWG Homo sapiens
-
CoA + Ac-MLGPEGGRWG
-
?
acetyl-CoA + peptide Homo sapiens
-
CoA + Nalpha-acetylpeptide
-
?

Organism

Organism UniProt Comment Textmining
Homo sapiens
-
-
-
Homo sapiens Q9GZZ1
-
-

Purification (Commentary)

Purification (Comment) Organism
HiTrap SP column chromatography, and Superdex 75 gel filtration Homo sapiens
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 Homo sapiens

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
acetyl-CoA + MLGPEGGRWG
-
Homo sapiens CoA + Ac-MLGPEGGRWG
-
?
acetyl-CoA + MLGPEGGRWGRPVGRRRRP
-
Homo sapiens acetyl-CoA + Nalpha-acetyl-MLGPEGGRWGRPVGRRRRP
-
?
acetyl-CoA + peptide
-
Homo sapiens CoA + Nalpha-acetylpeptide
-
?
acetyl-CoA + peptide peptide substrate binding structure, overview Homo sapiens CoA + Nalpha-acetylpeptide
-
?

Synonyms

Synonyms Comment Organism
Naa50p
-
Homo sapiens
Naa50p subunit of NatA Homo sapiens
Naa50p (NAT5/SAN) N-terminal acetyltransferase complex
-
Homo sapiens
NAT
-
Homo sapiens
NAT5/SAN
-
Homo sapiens
NatA
-
Homo sapiens

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
assay at Homo sapiens

Cofactor

Cofactor Comment Organism Structure
acetyl-CoA
-
Homo sapiens