Literature summary for 2.3.1.254 extracted from

Hong, H.; Cai, Y.; Zhang, S.; Ding, H.; Wang, H.; Han, A.
Molecular basis of substrate specific acetylation by N-terminal acetyltransferase NatB (2017), Structure, 25, 641-649.e3 .

Search for more literature for 2.3.1.254

Cloned(Commentary)

Cloned (Comment)	Organism
genes NAA20 and NAA25, sequence comparisons, functional recombinant coexpression of a full-length auxiliary subunit Naa25 subunit (residues 1-745) with a full-length (residues 1-188) or a deletion mutant (residues 1-170) of the catalytic subunit Naa20 as heterodimeric NatB holoenzymes in Escherichia coli, recombinant expression of His- and/or FLAG-tagged NatB wild-type and mutants enzymes in Escherichia coli	Candida albicans

Cloned (Comment)

Organism

genes NAA20 and NAA25, sequence comparisons, functional recombinant coexpression of a full-length auxiliary subunit Naa25 subunit (residues 1-745) with a full-length (residues 1-188) or a deletion mutant (residues 1-170) of the catalytic subunit Naa20 as heterodimeric NatB holoenzymes in Escherichia coli, recombinant expression of His- and/or FLAG-tagged NatB wild-type and mutants enzymes in Escherichia coli

Candida albicans

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified NatB free and complexed with bisubstrate inhibitor, NatB holoenzyme in the presence of CoA and substrate peptides, best crystals are grown in a condition containing 25 mM MES, pH 6.5, 5% PEG 600, 1.5% PEG 1000, 5% glycerol, 4% 1,3-butanediol, and 5 mM DTT, improved using the NatB complex with Naa20 (residue 1-170), best crystallization condition for NatB and the inhibitor complex is 20 mM Tris, pH 7.5, 20 mM NaCl, 2.2% PEG 4000, 5% glycerol, 4% 1,3-butanediol, and 5 mM DTT, 2-4 days at room temperature, X-ray diffraction structure determination and analysis at 2.33-2.75 A resolution	Candida albicans

Crystallization (Comment)

Organism

purified NatB free and complexed with bisubstrate inhibitor, NatB holoenzyme in the presence of CoA and substrate peptides, best crystals are grown in a condition containing 25 mM MES, pH 6.5, 5% PEG 600, 1.5% PEG 1000, 5% glycerol, 4% 1,3-butanediol, and 5 mM DTT, improved using the NatB complex with Naa20 (residue 1-170), best crystallization condition for NatB and the inhibitor complex is 20 mM Tris, pH 7.5, 20 mM NaCl, 2.2% PEG 4000, 5% glycerol, 4% 1,3-butanediol, and 5 mM DTT, 2-4 days at room temperature, X-ray diffraction structure determination and analysis at 2.33-2.75 A resolution

Candida albicans

Protein Variants

Protein Variants	Comment	Organism
A77S	site-directed mutagenesis of the Naa20 subunit, altered reaction kinetics compared to wild-type enzyme	Candida albicans
E25A	site-directed mutagenesis of the Naa20 subunit, altered reaction kinetics compared to wild-type enzyme	Candida albicans
F112A	site-directed mutagenesis of the Naa20 subunit, altered reaction kinetics compared to wild-type enzyme	Candida albicans
F112H	site-directed mutagenesis of the Naa20 subunit, altered reaction kinetics compared to wild-type enzyme	Candida albicans
F27A	site-directed mutagenesis of the Naa20 subunit, altered reaction kinetics compared to wild-type enzyme	Candida albicans
F27Y	site-directed mutagenesis of the Naa20 subunit, altered reaction kinetics compared to wild-type enzyme	Candida albicans
F490A/F493A	site-directed mutagenesis of the Naa25 subunit	Candida albicans
G140A	site-directed mutagenesis of the Naa20 subunit, altered reaction kinetics compared to wild-type enzyme	Candida albicans
H74A	site-directed mutagenesis of the Naa20 subunit, altered reaction kinetics compared to wild-type enzyme	Candida albicans
H74A/T76A	site-directed mutagenesis of the Naa20 subunit, altered reaction kinetics compared to wild-type enzyme	Candida albicans
L23A	site-directed mutagenesis of the Naa20 subunit, altered reaction kinetics compared to wild-type enzyme	Candida albicans
additional information	generation of a deletion mutant (residues 1-170) of the catalytic subunit Naa20. Structure of mutant NatB/Naa20 (residue 1-170) in complex with a peptide substrate MEAHNK-biotin and structure of NatB/Naa20 (full-length) in complex with a bisubstrate inhibitor CoA-MDSEVAALVID	Candida albicans
T24P	site-directed mutagenesis of the Naa20 subunit, altered reaction kinetics compared to wild-type enzyme	Candida albicans
T76A	site-directed mutagenesis of the Naa20 subunit, altered reaction kinetics compared to wild-type enzyme	Candida albicans
Y124F	site-directed mutagenesis of the Naa20 subunit, altered reaction kinetics compared to wild-type enzyme	Candida albicans
Y138A	site-directed mutagenesis of the Naa20 subunit, altered reaction kinetics compared to wild-type enzyme	Candida albicans
Y138A/Y139A	site-directed mutagenesis of the Naa20 subunit, altered reaction kinetics compared to wild-type enzyme	Candida albicans
Y138F	site-directed mutagenesis of the Naa20 subunit, altered reaction kinetics compared to wild-type enzyme	Candida albicans
Y139A	site-directed mutagenesis of the Naa20 subunit, altered reaction kinetics compared to wild-type enzyme	Candida albicans
Y139F	site-directed mutagenesis of the Naa20 subunit, altered reaction kinetics compared to wild-type enzyme	Candida albicans
Y362A	site-directed mutagenesis of the Naa25 subunit	Candida albicans
Y404A	site-directed mutagenesis of the Naa25 subunit	Candida albicans

Inhibitors

Inhibitors	Comment	Organism	Structure
CoA-MDSEVAALVID	a bisubstrate inhibitor	Candida albicans

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
acetyl-CoA + an N-terminal L-methionyl-L-asparaginyl-[protein]	Candida albicans	-	an N-terminal Nalpha-acetyl-L-methionyl-L-asparginyl-[protein] + CoA	-	?
acetyl-CoA + an N-terminal L-methionyl-L-asparaginyl-[protein]	Candida albicans ATCC MYA-2876	-	an N-terminal Nalpha-acetyl-L-methionyl-L-asparginyl-[protein] + CoA	-	?
acetyl-CoA + an N-terminal L-methionyl-L-aspartyl-[protein]	Candida albicans	-	an N-terminal Nalpha-acetyl-L-methionyl-L-aspartyl-[protein] + CoA	-	?
acetyl-CoA + an N-terminal L-methionyl-L-aspartyl-[protein]	Candida albicans ATCC MYA-2876	-	an N-terminal Nalpha-acetyl-L-methionyl-L-aspartyl-[protein] + CoA	-	?
acetyl-CoA + an N-terminal L-methionyl-L-glutaminyl-[protein]	Candida albicans	-	an N-terminal Nalpha-acetyl-L-methionyl-L-glutaminyl-[protein] + CoA	-	?
acetyl-CoA + an N-terminal L-methionyl-L-glutaminyl-[protein]	Candida albicans ATCC MYA-2876	-	an N-terminal Nalpha-acetyl-L-methionyl-L-glutaminyl-[protein] + CoA	-	?
acetyl-CoA + an N-terminal L-methionyl-L-glutamyl-[protein]	Candida albicans	-	an N-terminal Nalpha-acetyl-L-methionyl-L-glutamyl-[protein] + CoA	-	?
acetyl-CoA + an N-terminal L-methionyl-L-glutamyl-[protein]	Candida albicans ATCC MYA-2876	-	an N-terminal Nalpha-acetyl-L-methionyl-L-glutamyl-[protein] + CoA	-	?

Organism

Organism	UniProt	Comment	Textmining
Candida albicans	Q5AAR6 AND Q5AB99	subunits Naa20 and Naa25 of enzyme complex NatB	-
Candida albicans ATCC MYA-2876	Q5AAR6 AND Q5AB99	subunits Naa20 and Naa25 of enzyme complex NatB	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged wild-type and mutant NatB complexes from Escherichia coli by affinity chromatography, to homogeneity	Candida albicans

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
acetyl-CoA + an N-terminal L-methionyl-L-asparaginyl-[protein]	-	Candida albicans	an N-terminal Nalpha-acetyl-L-methionyl-L-asparginyl-[protein] + CoA	-	?
acetyl-CoA + an N-terminal L-methionyl-L-asparaginyl-[protein]	-	Candida albicans ATCC MYA-2876	an N-terminal Nalpha-acetyl-L-methionyl-L-asparginyl-[protein] + CoA	-	?
acetyl-CoA + an N-terminal L-methionyl-L-aspartyl-[protein]	-	Candida albicans	an N-terminal Nalpha-acetyl-L-methionyl-L-aspartyl-[protein] + CoA	-	?
acetyl-CoA + an N-terminal L-methionyl-L-aspartyl-[protein]	-	Candida albicans ATCC MYA-2876	an N-terminal Nalpha-acetyl-L-methionyl-L-aspartyl-[protein] + CoA	-	?
acetyl-CoA + an N-terminal L-methionyl-L-glutaminyl-[protein]	-	Candida albicans	an N-terminal Nalpha-acetyl-L-methionyl-L-glutaminyl-[protein] + CoA	-	?
acetyl-CoA + an N-terminal L-methionyl-L-glutaminyl-[protein]	-	Candida albicans ATCC MYA-2876	an N-terminal Nalpha-acetyl-L-methionyl-L-glutaminyl-[protein] + CoA	-	?
acetyl-CoA + an N-terminal L-methionyl-L-glutamyl-[protein]	-	Candida albicans	an N-terminal Nalpha-acetyl-L-methionyl-L-glutamyl-[protein] + CoA	-	?
acetyl-CoA + an N-terminal L-methionyl-L-glutamyl-[protein]	-	Candida albicans ATCC MYA-2876	an N-terminal Nalpha-acetyl-L-methionyl-L-glutamyl-[protein] + CoA	-	?
additional information	substrate specificity of NatB is determined by the first two amino acids of the substrate protein/peptide. The substrate's N-terminus is anchored into the NatB catalytic pocket by hydrogen bonds. The first two amino acids Met and Asp of a substrate peptide mediate the major interactions with the active site in the Naa20 subunit. The hydrogen bonds between the substrate Asp and pocket residues of Naa20 are essential to determine the NatB substrate specificity. A hydrogen bond between the amino group of the substrate Met and a carbonyl group in the Naa20 active site directly anchors the substrate toward acetyl-CoA. NatB has a unique substrate specificity different from all other NATs, which requires acidic amino acids or their amides at the second position. No activity with the NatC substrate MLRFVTANSQDNGRPVGRK and with the NatA substrate SASEAG	Candida albicans	?	-	-
additional information	substrate specificity of NatB is determined by the first two amino acids of the substrate protein/peptide. The substrate's N-terminus is anchored into the NatB catalytic pocket by hydrogen bonds. The first two amino acids Met and Asp of a substrate peptide mediate the major interactions with the active site in the Naa20 subunit. The hydrogen bonds between the substrate Asp and pocket residues of Naa20 are essential to determine the NatB substrate specificity. A hydrogen bond between the amino group of the substrate Met and a carbonyl group in the Naa20 active site directly anchors the substrate toward acetyl-CoA. NatB has a unique substrate specificity different from all other NATs, which requires acidic amino acids or their amides at the second position. No activity with the NatC substrate MLRFVTANSQDNGRPVGRK and with the NatA substrate SASEAG	Candida albicans ATCC MYA-2876	?	-	-

Subunits

Subunits	Comment	Organism
More	the auxiliary subunit Naa25 of NatB forms a horseshoe-like deck to hold specifically its catalytic subunit Naa20. Analysis of the structure of mutant NatB/Naa20(residue 1-170) in complex with a peptide substrate MEAHNK-biotin and of the structure of NatB/Naa20(full-length) in complex with a bisubstrate inhibitor CoA-MDSEVAALVID, structure comparisons, overview	Candida albicans

Synonyms

Synonyms	Comment	Organism
N-terminal acetyltransferase	-	Candida albicans
NAA20	-	Candida albicans
NAA25	-	Candida albicans
natB	-	Candida albicans

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	assay at	Candida albicans

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8	-	assay at	Candida albicans

Cofactor

Cofactor	Comment	Organism	Structure
acetyl-CoA	-	Candida albicans

General Information

General Information	Comment	Organism
evolution	the family of N-terminal acetyltransferases (NATs) has six subtypes with their unique substrate specificity, NatA-NatF. The substrate specificity of Nats is determined by the first two amino acids of the substrate protein/peptide. NatB has a unique substrate specificity different from all other NATs, which requires acidic amino acids or their amides at the second position	Candida albicans
additional information	the auxiliary subunit Naa25 of NatB forms a horseshoe-like deck to hold specifically its catalytic subunit Naa20. The first two amino acids Met and Asp of a substrate peptide mediate the major interactions with the active site in the Naa20 subunit. The hydrogen bonds between the substrate Asp and pocket residues of Naa20 are essential to determine the NatB substrate specificity. A hydrogen bond between the amino group of the substrate Met and a carbonyl group in the Naa20 active site directly anchors the substrate toward acetyl-CoA Unique molecular mechanism of specific N-terminal acetylation acted by NatB, substrate recognition and acetylation of NatB, overview	Candida albicans