Any feedback?
Please rate this page
(literature.php)
(0/150)

BRENDA support

Literature summary 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

Activating Compound

EC Number Activating Compound Comment Organism Structure
3.1.1.1 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)

EC Number Cloned (Comment) Organism
3.1.1.1 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
3.1.1.1 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)

EC Number Crystallization (Comment) Organism
3.1.1.1 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

EC Number Protein Variants Comment Organism
3.1.1.1 F210L resulting from point mutations T634C, site-directed mutagenesis, the mutant shows lower thermostability compared to double mutant N96S/F210L uncultured bacterium
3.1.1.1 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
3.1.1.1 N96S resulting from point mutations A293G, site-directed mutagenesis, the mutant shows lower thermostability compared to double mutant N96S/F210L uncultured bacterium
3.1.1.1 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

EC Number General Stability Organism
3.1.1.1 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

EC Number Inhibitors Comment Organism Structure
3.1.1.1 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]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.1.1.1 0.1 0.58 4-nitrophenyl acetate recombinant wild-type enzyme, pH 8.5, 30°C uncultured bacterium
3.1.1.1 0.13
-
 4-nitrophenyl butyrate recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium
3.1.1.1 0.19
-
 4-nitrophenyl octanoate recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium
3.1.1.1 0.2
-
 2-naphthyl acetate recombinant wild-type enzyme, pH 8.5, 30°C uncultured bacterium
3.1.1.1 0.41
-
 2-naphthyl acetate recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium
3.1.1.1 0.42
-
 4-nitrophenyl acetate recombinant mutant N96S, pH 8.5, 30°C uncultured bacterium
3.1.1.1 0.46
-
 7-aminocephalosporanic acid recombinant wild-type enzyme, pH 8.5, 30°C uncultured bacterium
3.1.1.1 0.47
-
 4-methylumbelliferyl acetate recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium
3.1.1.1 0.51
-
 7-aminocephalosporanic acid recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium
3.1.1.1 0.63
-
 4-nitrophenyl acetate recombinant mutant F210L, pH 8.5, 30°C uncultured bacterium
3.1.1.1 0.7
-
 4-nitrophenyl butyrate recombinant wild-type enzyme, pH 8.5, 30°C uncultured bacterium
3.1.1.1 0.76
-
 4-nitrophenyl acetate recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium
3.1.1.1 166.7
-
 4-methylumbelliferyl acetate recombinant wild-type enzyme, pH 8.5, 30°C uncultured bacterium

Localization

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

Metals/Ions

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

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
3.1.1.1 216000
-
 recombinant wild-type enzyme, gel filtration uncultured bacterium

Organism

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

Purification (Commentary)

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

Source Tissue

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

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
3.1.1.1 1.86
-
 purified recombinant mutant NaM1H2, pH 8.5, 30°C, substrate 4-nitrophenyl octanoate uncultured bacterium
3.1.1.1 6.05
-
 purified recombinant wild-type enzyme, pH 8.5, 30°C, substrate xylan uncultured bacterium
3.1.1.1 12.96
-
 purified recombinant wild-type enzyme, pH 8.5, 30°C, substrate 4-nitrophenyl butyrate uncultured bacterium
3.1.1.1 20.46
-
 purified recombinant mutant NaM1H2, pH 8.5, 30°C, substrate 4-nitrophenyl butyrate uncultured bacterium
3.1.1.1 78.51
-
 purified recombinant mutant NaM1H2, pH 8.5, 30°C, substrate 2-naphthyl acetate uncultured bacterium
3.1.1.1 106.25
-
 purified recombinant mutant NaM1H2, pH 8.5, 30°C, substrate 7-aminocephalosporanic acid uncultured bacterium
3.1.1.1 162.2
-
 purified recombinant mutant F210L, pH 8.5, 30°C, substrate 4-nitrophenyl acetate uncultured bacterium
3.1.1.1 200
-
 purified recombinant wild-type enzyme, pH 8.5, 30°C, substrate 7-aminocephalosporanic acid uncultured bacterium
3.1.1.1 211.7
-
 purified recombinant mutant NaM1H2, pH 8.5, 30°C, substrate 4-methylumbelliferyl acetate uncultured bacterium
3.1.1.1 222.2
-
 purified recombinant wild-type enzyme, pH 8.5, 30°C, substrate 2-naphthyl acetate uncultured bacterium
3.1.1.1 277.8
-
 purified recombinant wild-type enzyme, pH 8.5, 30°C, substrate 4-methylumbelliferyl acetate uncultured bacterium
3.1.1.1 348.3
-
 purified recombinant mutant NaM1H2, pH 8.5, 30°C, substrate 4-nitrophenyl acetate uncultured bacterium
3.1.1.1 488.9 832.4 purified recombinant wild-type enzyme, pH 8.5, 30°C, substrate 4-nitrophenyl acetate uncultured bacterium
3.1.1.1 955.4
-
 purified recombinant mutant N96S, pH 8.5, 30°C, substrate 4-nitrophenyl acetate uncultured bacterium

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.1.1.1 2-naphthyl acetate + H2O
-
 uncultured bacterium 2-naphthol + acetate
-
 ?
3.1.1.1 4-methylumbelliferyl acetate + H2O
-
 uncultured bacterium 4-methylumbelliferol + acetate
-
 ?
3.1.1.1 4-nitrophenyl acetate + H2O best substrate uncultured bacterium 4-nitrophenol + acetate
-
 ?
3.1.1.1 4-nitrophenyl butyrate + H2O
-
 uncultured bacterium 4-nitrophenol + butyrate
-
 ?
3.1.1.1 4-nitrophenyl octanoate + H2O low activity by mutant NaM1H2, not by wild-type enzyme uncultured bacterium 4-nitrophenol + octanoate
-
 ?
3.1.1.1 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
-
 ?
3.1.1.1 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 ?
-
 ?
3.1.1.1 additional information in general, CE7 enzymes possess a unique and narrow specificity for acetylated substrates. Enzyme NaM2 shows no tributyrin-hydrolyzing activity uncultured bacterium ?
-
 ?
3.1.1.1 additional information in general, CE7 enzymes possess a unique and narrow specificity for acetylated substrates. Enzyme NaM3 shows no tributyrin-hydrolyzing activity uncultured bacterium ?
-
 ?
3.1.1.1 xylan + H2O polymeric xylan, low activity by the wild-type enzyme, no activity by enzyme mutant NaM1H2 uncultured bacterium ?
-
 ?

Subunits

EC Number Subunits Comment Organism
3.1.1.1 ? x * 26000, about, sequence calculation uncultured bacterium
3.1.1.1 ? x * 35900, about, sequence calculation uncultured bacterium
3.1.1.1 homohexamer 6 * 35600, about, sequence calculation uncultured bacterium
3.1.1.1 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

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

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.1.1.1 30
-
 recombinant wild-type enzyme NaM1 uncultured bacterium
3.1.1.1 35
-
 recombinant wild-type enzyme NaM1 in presence of 1 M NaCl uncultured bacterium
3.1.1.1 40
-
 recombinant enzyme mutant NaM1H2 uncultured bacterium

Temperature Range [°C]

EC Number Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
3.1.1.1 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
3.1.1.1 25 75 recombinant mutant NaM1H2, activity range, profile overview uncultured bacterium

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
3.1.1.1 additional information
-
 a reduced side-chain volume of protein core residues is relevant to the thermal stability of NaM1 uncultured bacterium
3.1.1.1 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
3.1.1.1 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
3.1.1.1 55
-
 purified recombinant wild-type enzyme, inactivation after 20 min uncultured bacterium
3.1.1.1 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
3.1.1.1 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
3.1.1.1 70
-
 purified recombinant mutant NaM1H2, inactivation within 5 min uncultured bacterium

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.1.1.1 1.11
-
 4-nitrophenyl octanoate recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium
3.1.1.1 7.67
-
 4-nitrophenyl butyrate recombinant wild-type enzyme, pH 8.5, 30°C uncultured bacterium
3.1.1.1 12.18
-
 4-nitrophenyl butyrate recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium
3.1.1.1 46.7
-
 7-aminocephalosporanic acid recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium
3.1.1.1 46.7
-
 2-naphthyl acetate recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium
3.1.1.1 95.9
-
 4-nitrophenyl acetate recombinant mutant F210L, pH 8.5, 30°C uncultured bacterium
3.1.1.1 126
-
 4-methylumbelliferyl acetate recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium
3.1.1.1 133.3
-
 7-aminocephalosporanic acid recombinant wild-type enzyme, pH 8.5, 30°C uncultured bacterium
3.1.1.1 133.3
-
 2-naphthyl acetate recombinant wild-type enzyme, pH 8.5, 30°C uncultured bacterium
3.1.1.1 166.7
-
 4-methylumbelliferyl acetate recombinant wild-type enzyme, pH 8.5, 30°C uncultured bacterium
3.1.1.1 206
-
 4-nitrophenyl acetate recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium
3.1.1.1 293.3 492.3 4-nitrophenyl acetate recombinant wild-type enzyme, pH 8.5, 30°C uncultured bacterium
3.1.1.1 565
-
 4-nitrophenyl acetate recombinant mutant N96S, pH 8.5, 30°C uncultured bacterium

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.1.1.1 8.5
-
 recombinant wild-type enzyme uncultured bacterium

pH Stability

EC Number pH Stability pH Stability Maximum Comment Organism
3.1.1.1 8
-
 purified recombinant wild-type enzyme, most stable at uncultured bacterium

pI Value

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

General Information

EC Number General Information Comment Organism
3.1.1.1 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
3.1.1.1 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
3.1.1.1 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
3.1.1.1 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
3.1.1.1 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
3.1.1.1 additional information the NaM3 sequence contains catalytic residues typical of SGNH hydrolases: Ser49, Gly106, Asn163, and His225 uncultured bacterium

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
3.1.1.1 5.83
-
 4-nitrophenyl octanoate recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium
3.1.1.1 11
-
 4-nitrophenyl butyrate recombinant wild-type enzyme, pH 8.5, 30°C uncultured bacterium
3.1.1.1 114
-
 2-naphthyl acetate recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium
3.1.1.1 122
-
 7-aminocephalosporanic acid recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium
3.1.1.1 167
-
 4-methylumbelliferyl acetate recombinant wild-type enzyme, pH 8.5, 30°C uncultured bacterium
3.1.1.1 260
-
 7-aminocephalosporanic acid recombinant wild-type enzyme, pH 8.5, 30°C uncultured bacterium
3.1.1.1 272 2720 4-nitrophenyl acetate recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium
3.1.1.1 667
-
 2-naphthyl acetate recombinant wild-type enzyme, pH 8.5, 30°C uncultured bacterium
3.1.1.1 843 3260 4-nitrophenyl acetate recombinant wild-type enzyme, pH 8.5, 30°C uncultured bacterium
3.1.1.1 1280
-
 4-nitrophenyl butyrate recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium
3.1.1.1 1340
-
 4-nitrophenyl acetate recombinant mutant N96S, pH 8.5, 30°C uncultured bacterium
3.1.1.1 1520
-
 4-nitrophenyl acetate recombinant mutant F210L, pH 8.5, 30°C uncultured bacterium
3.1.1.1 2680
-
 4-methylumbelliferyl acetate recombinant mutant NaM1H2, pH 8.5, 30°C uncultured bacterium