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 (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 | 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 | Comment | Organism | Structure |
---|---|---|---|
CoA-MDSEVAALVID | a bisubstrate inhibitor | Candida albicans |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
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 | 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 (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant NatB complexes from Escherichia coli by affinity chromatography, to homogeneity | Candida albicans |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
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 | 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 | Comment | Organism |
---|---|---|
N-terminal acetyltransferase | - |
Candida albicans |
NAA20 | - |
Candida albicans |
NAA25 | - |
Candida albicans |
natB | - |
Candida albicans |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Candida albicans |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Candida albicans |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
acetyl-CoA | - |
Candida albicans |
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 |