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Literature summary for 3.1.1.1 extracted from

  • Adesioye, F.A.; Makhalanyane, T.P.; Vikram, S.; Sewell, B.T.; Schubert, W.D.; Cowan, D.A.
    Structural characterization and directed evolution of a novel acetyl xylan esterase reveals thermostability determinants of the carbohydrate esterase 7 family (2018), Appl. Environ. Microbiol., 84, e02695-17 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 3.1.1.1

Activating Compound

Activating Compound Comment Organism Structure
additional information NaM1 activity and thermal stability are not affected by bovine serum albumin or other stabilizing solutes other than NaCl and trehalose uncultured bacterium

Cloned(Commentary)

Cloned (Comment) Organism
synthetic construct NaM1, cloned from Namib soil hypolith metagenomic data set, i.e. hypolithome, DNA and amino acid sequence determination and analysis, sequence comparisons and phylogenetic analysis, recombinant expression of N-terminally His6-tagged enzyme in Escherichia coli strain BL21(DE3) uncultured bacterium
synthetic construct NaM2, cloned from Namib soil hypolith metagenomic data set, i.e. hypolithome, DNA and amino acid sequence determination and analysis, sequence comparisons and phylogenetic analysis, recombinant expression of N-terminally His6-tagged enzyme in Escherichia coli strain BL21(DE3) uncultured bacterium

Crystallization (Commentary)

Crystallization (Comment) Organism
purified recombinant wild-type enzyme NaM1, from 0.1 M MES, pH 8.5, with 25% w/v PEG 8000, X-ray diffraction structure determination and analysis at 2.03 A resolution, molecular replacement uncultured bacterium

Protein Variants

Protein Variants Comment Organism
F210L resulting from point mutations T634C, site-directed mutagenesis, the mutant shows lower thermostability compared to double mutant N96S/F210L uncultured bacterium
additional information error-prone PCR and site-directed mutagenesis for directed evolution experiments increasing the enzyme's thermal stability and catalytic efficiency, overview. Enzyme variants NaM1H2, NaM1D8, and NaM1B4 each containing two point mutations, one of which is silent in both NaM1D8 and NaM1B4. NaM1H2 has two amino acid substitutions, N96S and F210L uncultured bacterium
N96S resulting from point mutations A293G, site-directed mutagenesis, the mutant shows lower thermostability compared to double mutant N96S/F210L uncultured bacterium
N96S/F210L mutant NaM1H2, site-directed mutagenesis, the mutation results in a simultaneous improvement in thermal stability and catalytic efficiency and increases the acyl moiety substrate range of enzyme mutant NaM1H2 uncultured bacterium

General Stability

General Stability Organism
wild-type NaM1 activity and thermal stability are not affected by bovine serum albumin or other stabilizing solutes other than NaCl and trehalose uncultured bacterium

Inhibitors

Inhibitors Comment Organism Structure
additional information NaM1 activity and thermal stability are not affected by bovine serum albumin or other stabilizing solutes other than NaCl and trehalose uncultured bacterium

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.1 0.58 4-nitrophenyl acetate recombinant wild-type enzyme, pH 8.5, 30°C uncultured bacterium
0.13
-
 4-nitrophenyl butyrate recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium
0.19
-
 4-nitrophenyl octanoate recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium
0.2
-
 2-naphthyl acetate recombinant wild-type enzyme, pH 8.5, 30°C uncultured bacterium
0.41
-
 2-naphthyl acetate recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium
0.42
-
 4-nitrophenyl acetate recombinant mutant N96S, pH 8.5, 30°C uncultured bacterium
0.46
-
 7-aminocephalosporanic acid recombinant wild-type enzyme, pH 8.5, 30°C uncultured bacterium
0.47
-
 4-methylumbelliferyl acetate recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium
0.51
-
 7-aminocephalosporanic acid recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium
0.63
-
 4-nitrophenyl acetate recombinant mutant F210L, pH 8.5, 30°C uncultured bacterium
0.7
-
 4-nitrophenyl butyrate recombinant wild-type enzyme, pH 8.5, 30°C uncultured bacterium
0.76
-
 4-nitrophenyl acetate recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium
166.7
-
 4-methylumbelliferyl acetate recombinant wild-type enzyme, pH 8.5, 30°C uncultured bacterium

Localization

Localization Comment Organism GeneOntology No. Textmining
extracellular residues 1 to 32 encoded by the NaM3 gene represent a signal peptide uncultured bacterium
-
-
intracellular
-
 uncultured bacterium 5622
-
additional information no signal peptide sequences identified for NaM1 uncultured bacterium
-
-
additional information no signal peptide sequences identified for NaM2 uncultured bacterium
-
-

Metals/Ions

Metals/Ions Comment Organism Structure
NaCl enzyme NaM1 reatins about 50% activity in 5 M NaCl at 40°C after 1 h uncultured bacterium

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
216000
-
 recombinant wild-type enzyme, gel filtration uncultured bacterium

Organism

Organism UniProt Comment Textmining
uncultured bacterium
-
 gene from Namib hypolith metagenomic data set
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His6-tagged enzyme from Escherichia coli strain BL21(DE3) by cobalt affinity chromatography to over 95% purity uncultured bacterium

Source Tissue

Source Tissue Comment Organism Textmining
additional information optimal production at 18°C for NaM3 uncultured bacterium
-
additional information optimal production at 25°C for NaM1 uncultured bacterium
-
additional information optimal production at 25°C for NaM2 uncultured bacterium
-

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.86
-
 purified recombinant mutant NaM1H2, pH 8.5, 30°C, substrate 4-nitrophenyl octanoate uncultured bacterium
6.05
-
 purified recombinant wild-type enzyme, pH 8.5, 30°C, substrate xylan uncultured bacterium
12.96
-
 purified recombinant wild-type enzyme, pH 8.5, 30°C, substrate 4-nitrophenyl butyrate uncultured bacterium
20.46
-
 purified recombinant mutant NaM1H2, pH 8.5, 30°C, substrate 4-nitrophenyl butyrate uncultured bacterium
78.51
-
 purified recombinant mutant NaM1H2, pH 8.5, 30°C, substrate 2-naphthyl acetate uncultured bacterium
106.25
-
 purified recombinant mutant NaM1H2, pH 8.5, 30°C, substrate 7-aminocephalosporanic acid uncultured bacterium
162.2
-
 purified recombinant mutant F210L, pH 8.5, 30°C, substrate 4-nitrophenyl acetate uncultured bacterium
200
-
 purified recombinant wild-type enzyme, pH 8.5, 30°C, substrate 7-aminocephalosporanic acid uncultured bacterium
211.7
-
 purified recombinant mutant NaM1H2, pH 8.5, 30°C, substrate 4-methylumbelliferyl acetate uncultured bacterium
222.2
-
 purified recombinant wild-type enzyme, pH 8.5, 30°C, substrate 2-naphthyl acetate uncultured bacterium
277.8
-
 purified recombinant wild-type enzyme, pH 8.5, 30°C, substrate 4-methylumbelliferyl acetate uncultured bacterium
348.3
-
 purified recombinant mutant NaM1H2, pH 8.5, 30°C, substrate 4-nitrophenyl acetate uncultured bacterium
488.9 832.4 purified recombinant wild-type enzyme, pH 8.5, 30°C, substrate 4-nitrophenyl acetate uncultured bacterium
955.4
-
 purified recombinant mutant N96S, pH 8.5, 30°C, substrate 4-nitrophenyl acetate uncultured bacterium

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2-naphthyl acetate + H2O
-
 uncultured bacterium 2-naphthol + acetate
-
 ?
4-methylumbelliferyl acetate + H2O
-
 uncultured bacterium 4-methylumbelliferol + acetate
-
 ?
4-nitrophenyl acetate + H2O best substrate uncultured bacterium 4-nitrophenol + acetate
-
 ?
4-nitrophenyl butyrate + H2O
-
 uncultured bacterium 4-nitrophenol + butyrate
-
 ?
4-nitrophenyl octanoate + H2O low activity by mutant NaM1H2, not by wild-type enzyme uncultured bacterium 4-nitrophenol + octanoate
-
 ?
7-aminocephalosporanic acid + H2O
-
 uncultured bacterium (6R,7R)-7-amino-3-(hydroxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid + acetate
-
 ?
additional information CE7 enzymes, like mesophilic CE7 AcXE, Axe1NaM1, possess a unique and narrow specificity for acetylated substrates. Enzyme NaM1 also shows tributyrin-hydrolyzing activity. No activity of the wild-type enzyme with 4-nitrophenol octanoate and 4-nitrophenol palmitate, mutant NaM1H2 shows low activity with 4-nitrophenol octanoate uncultured bacterium ?
-
 ?
additional information in general, CE7 enzymes possess a unique and narrow specificity for acetylated substrates. Enzyme NaM2 shows no tributyrin-hydrolyzing activity uncultured bacterium ?
-
 ?
additional information in general, CE7 enzymes possess a unique and narrow specificity for acetylated substrates. Enzyme NaM3 shows no tributyrin-hydrolyzing activity uncultured bacterium ?
-
 ?
xylan + H2O polymeric xylan, low activity by the wild-type enzyme, no activity by enzyme mutant NaM1H2 uncultured bacterium ?
-
 ?

Subunits

Subunits Comment Organism
? x * 26000, about, sequence calculation uncultured bacterium
? x * 35900, about, sequence calculation uncultured bacterium
homohexamer 6 * 35600, about, sequence calculation uncultured bacterium
More the hexamer is composed of two trimers, each NaM1 monomer directly interacts with the two monomers from the same trimer and with two monomers from the opposite trimer uncultured bacterium

Synonyms

Synonyms Comment Organism
acetyl xylan esterase
-
 uncultured bacterium
AcXE
-
 uncultured bacterium
Axe1NaM1
-
 uncultured bacterium
Axe1NaM2
-
 uncultured bacterium
Axe1NaM3
-
 uncultured bacterium
CE7 AcXE
-
 uncultured bacterium
NaM1
-
 uncultured bacterium
NaM2
-
 uncultured bacterium
NaM3
-
 uncultured bacterium

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
 recombinant wild-type enzyme NaM1 uncultured bacterium
35
-
 recombinant wild-type enzyme NaM1 in presence of 1 M NaCl uncultured bacterium
40
-
 recombinant enzyme mutant NaM1H2 uncultured bacterium

Temperature Range [°C]

Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
20 50 recombinant wild-type enzyme, activity range, profile overview. Most stable at 30°C, 10% activity at 40°C after 1 h, 33% activity at 50°C with 1 M NaCl after 1 h, loss of activity at 55°C after 1 h uncultured bacterium
25 75 recombinant mutant NaM1H2, activity range, profile overview uncultured bacterium

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
additional information
-
 a reduced side-chain volume of protein core residues is relevant to the thermal stability of NaM1 uncultured bacterium
47 70 the wild-type enzyme has a melting temperature (Tm) of 47°C and an unfolding transition at 45°C, while the double mutant NaM1H2 melts at 63°C and unfolds at 60°C. The mutants NaM1N96S and NaM1F210L show thermal unfolding at 50°C and have a melting temperature of 52°C, and lose all secondary structures at 70°C uncultured bacterium
50
-
 purified recombinant wild-type enzyme lose of 50% activity after 25 min, mutant N96S loses 50% activity after 20 min, mutant F210L loses 80% after 20 min, while mutant NaM1H2 (N96S/F210L) is completely stable for 25 min uncultured bacterium
55
-
 purified recombinant wild-type enzyme, inactivation after 20 min uncultured bacterium
60
-
 purified recombinant wild-type enzyme, inactivation within 5 min. Purified recombinant mutant NaM1H2 is completely stable for 60 min, and shows 150% activity after 15 min uncultured bacterium
65
-
 purified recombinant mutant NaM1H2, completely stable for 10 min, loss of 30% activity after 15 min, of 50% after 25 min, and of 65% after 30 min uncultured bacterium
70
-
 purified recombinant mutant NaM1H2, inactivation within 5 min uncultured bacterium

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.11
-
 4-nitrophenyl octanoate recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium
7.67
-
 4-nitrophenyl butyrate recombinant wild-type enzyme, pH 8.5, 30°C uncultured bacterium
12.18
-
 4-nitrophenyl butyrate recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium
46.7
-
 7-aminocephalosporanic acid recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium
46.7
-
 2-naphthyl acetate recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium
95.9
-
 4-nitrophenyl acetate recombinant mutant F210L, pH 8.5, 30°C uncultured bacterium
126
-
 4-methylumbelliferyl acetate recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium
133.3
-
 7-aminocephalosporanic acid recombinant wild-type enzyme, pH 8.5, 30°C uncultured bacterium
133.3
-
 2-naphthyl acetate recombinant wild-type enzyme, pH 8.5, 30°C uncultured bacterium
166.7
-
 4-methylumbelliferyl acetate recombinant wild-type enzyme, pH 8.5, 30°C uncultured bacterium
206
-
 4-nitrophenyl acetate recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium
293.3 492.3 4-nitrophenyl acetate recombinant wild-type enzyme, pH 8.5, 30°C uncultured bacterium
565
-
 4-nitrophenyl acetate recombinant mutant N96S, pH 8.5, 30°C uncultured bacterium

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8.5
-
 recombinant wild-type enzyme uncultured bacterium

pH Stability

pH Stability pH Stability Maximum Comment Organism
8
-
 purified recombinant wild-type enzyme, most stable at uncultured bacterium

pI Value

Organism Comment pI Value Maximum pI Value
uncultured bacterium sequence calculation
-
 5.1
uncultured bacterium sequence calculation
-
 5.5
uncultured bacterium sequence calculation
-
 5.8

General Information

General Information Comment Organism
evolution the enzyme NaM1 belongs to the carbohydrate esterase 7 (CE7) family enzymes, i.e. Axe1NaM1. CE7 enzymes are intracellular enzymes that possess a unique and narrow specificity for acetylated substrates. The putative AcXE-encoding genes, Axe1NaM1 (CE7), Axe1NaM2 (CE7), and XynBNaM3-like (CE3), have 64, 69, and 59% sequence identities, respectively, to known AcXE sequences from actinobacteria and distinct domain arrangements. The Axe1 domains are encoded by nucleotides 19 to 966 and 19 to 972 of the Axe1NaM1 and Axe1NaM2 genes, respectively, and shared 46.5% sequence identity. The two genes are located on two distinct contigs. The Axe1NaM1, Axe1NaM2, and XynBNaM3-like proteins, i.e. NaM1, NaM2, and NaM3, all exhibit a Ser-His-Asp(Glu) catalytic triad and a GXSXG or GDS(L) motif typical of the CE7 or CE3 family, respectively uncultured bacterium
evolution the enzyme NaM2 belongs to the carbohydrate esterase 7 (CE7) family enzymes. CE7 enzymes are intracellular enzymes that possess a unique and narrow specificity for acetylated substrates. The putative AcXE-encoding genes, Axe1NaM1 (CE7), Axe1NaM2 (CE7), and XynBNaM3-like (CE3), have 64, 69, and 59% sequence identities, respectively, to known AcXE sequences from actinobacteria and distinct domain arrangements. The Axe1 domains are encoded by nucleotides 19 to 966 and 19 to 972 of the Axe1NaM1 and Axe1NaM2 genes, respectively, and share 46.5% sequence identity. The two genes are located on two distinct contigs. The Axe1NaM1, Axe1NaM2, and XynBNaM3-like proteins, i.e. NaM1, NaM2, and NaM3, all exhibit a Ser-His-Asp(Glu) catalytic triad and a GXSXG or GDS(L) motif typical of the CE7 or CE3 family, respectively uncultured bacterium
evolution the putative AcXE-encoding genes, Axe1NaM1 (CE7), Axe1NaM2 (CE7), and XynBNaM3-like (CE3), have 64, 69, and 59% sequence identities, respectively, to known AcXE sequences from actinobacteria and distinct domain arrangements. The Axe1 domains are encoded by nucleotides 19 to 966 and 19 to 972 of the Axe1NaM1 and Axe1NaM2 genes, respectively, and shared 46.5% sequence identity. The two genes are located on two distinct contigs. The Axe1NaM1, Axe1NaM2, and XynBNaM3-like proteins, i.e. NaM1, NaM2, and NaM3, all exhibit a Ser-His-Asp(Glu) catalytic triad and a GXSXG or GDS(L) motif typical of the CE7 or CE3 family, respectively uncultured bacterium
additional information catalytic residues of NaM1 correspond to Ser185, His304, and Asp275. Ser185 is located toward the end of a concave substrate binding pocket that extends to the S2 pocket accommodating the substrate acyl moiety. His304 bridges Ser185 and Asp275. In the presence of substrate, Asp275 polarizes His304 to deprotonate Ser185, allowing the latter to attack the carbonyl carbon of the substrate ester to initiate bond cleavage. Enzyme NaM1 exhibits the alpha/beta-hydrolase motif GXSXG (GYSQG) in loop beta6-alpha5. Structure-function analysis, overview uncultured bacterium
additional information catalytic residues of NaM1 correspond to Ser187, His307, and Asp273. Ser187 is located toward the end of a concave substrate binding pocket that extends to the S2 pocket accommodating the substrate acyl moiety. His307 bridges Ser185 and Asp273. In the presence of substrate, Asp273 polarizes His307 to deprotonate Ser187, allowing the latter to attack the carbonyl carbon of the substrate ester to initiate bond cleavage. Enzyme NaM1 exhibits the alpha/beta-hydrolase motif GXSXG (GYSQG) in loop beta6-alpha5. Structure-function analysis, overview uncultured bacterium
additional information the NaM3 sequence contains catalytic residues typical of SGNH hydrolases: Ser49, Gly106, Asn163, and His225 uncultured bacterium

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
5.83
-
 4-nitrophenyl octanoate recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium
11
-
 4-nitrophenyl butyrate recombinant wild-type enzyme, pH 8.5, 30°C uncultured bacterium
114
-
 2-naphthyl acetate recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium
122
-
 7-aminocephalosporanic acid recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium
167
-
 4-methylumbelliferyl acetate recombinant wild-type enzyme, pH 8.5, 30°C uncultured bacterium
260
-
 7-aminocephalosporanic acid recombinant wild-type enzyme, pH 8.5, 30°C uncultured bacterium
272 2720 4-nitrophenyl acetate recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium
667
-
 2-naphthyl acetate recombinant wild-type enzyme, pH 8.5, 30°C uncultured bacterium
843 3260 4-nitrophenyl acetate recombinant wild-type enzyme, pH 8.5, 30°C uncultured bacterium
1280
-
 4-nitrophenyl butyrate recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium
1340
-
 4-nitrophenyl acetate recombinant mutant N96S, pH 8.5, 30°C uncultured bacterium
1520
-
 4-nitrophenyl acetate recombinant mutant F210L, pH 8.5, 30°C uncultured bacterium
2680
-
 4-methylumbelliferyl acetate recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium