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Literature summary for 3.2.1.52 extracted from

  • Sumida, T.; Ishii, R.; Yanagisawa, T.; Yokoyama, S.; Ito, M.
    Molecular cloning and crystal structural analysis of a novel beta-N-acetylhexosaminidase from Paenibacillus sp. TS12 capable of degrading glycosphingolipids (2009), J. Mol. Biol., 392, 87-99.
    View publication on PubMed
Search for more literature for 3.2.1.52

Cloned(Commentary)

Cloned (Comment) Organism
expression of His-tagged wild-type isozymes Hex1 and Hex2, and of Hex1 mutants in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain DH5alpha Paenibacillus sp. TS12

Crystallization (Commentary)

Crystallization (Comment) Organism
purified recombinant wild-type isozymes Hex1 and Hex2, and mutant Hex1-DELTAC, and of isozymes in complex with N-acetyl-beta-D-glucosamine and N-acetyl-beta-D-galactosmaine, by hanging drop vapor diffusion method, mixing 0.001 ml of protein solution, with 10 mg/ml protein in 10 mM HEPES-sodium buffer, pH 7.4, 150 mM NaCl, 10 mM 2-mercaptoethanol, with 0.001 ml of precipitant containing 0.2 M (NH4)2SO4, 20% w/v PEG 2000 and 0.1 M sodium acetate buffer, pH 4.6, X-ray diffraction structure determination and analysis at 1.6-1.9 A resolution, molecular replacement method Paenibacillus sp. TS12

Protein Variants

Protein Variants Comment Organism
D321E site-directed mutagenesis, the Hex1 mutant shows reduced activity compared to the wild-type enzyme Paenibacillus sp. TS12
D321N site-directed mutagenesis, almost inactive Hex1 mutant Paenibacillus sp. TS12
E322D site-directed mutagenesis, the Hex1 mutant shows highly reduced activity compared to the wild-type enzyme Paenibacillus sp. TS12
E322Q site-directed mutagenesis, the Hex1 mutant shows reduced activity compared to the wild-type enzyme Paenibacillus sp. TS12
additional information construction of a C-terminally truncated TS12 beta-HEX1 mutant, Hex1-DELTAC, comprising amino acids 1-502. Hex1-DELTAC consists of two domains, an N-terminal domain, residues 14-157, that consists of two long alpha-helices and seven beta-sheets, and a central catalytic domain, the catalytic domain is a typical (beta/alpha)8-barrel, a cyclic 8fold repeat of beta-strand/loop/alpha-helix units in which the beta-strands form the central 8-stranded beta-barrel Paenibacillus sp. TS12

Inhibitors

Inhibitors Comment Organism Structure
Ca2+ slight inhibition at 5 mM Paenibacillus sp. TS12
Cu2+ strong inhibition at 5 mM Paenibacillus sp. TS12
Hg2+ strong inhibition at 5 mM Paenibacillus sp. TS12
K+ slight inhibition at 5 mM Paenibacillus sp. TS12
Mg2+ slight inhibition at 5 mM Paenibacillus sp. TS12
Na+ slight inhibition at 5 mM Paenibacillus sp. TS12
Ni2+ strong inhibition at 5 mM Paenibacillus sp. TS12

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.026
-
 4-nitrophenyl N-acetyl-beta-D-glucosamine pH 6.0, 37°C, mutant E322Q isozyme Hex1 Paenibacillus sp. TS12
0.049
-
 4-nitrophenyl N-acetyl-beta-D-glucosamine pH 6.0, 37°C, mutant E322D isozyme Hex1 Paenibacillus sp. TS12
0.073
-
 4-nitrophenyl N-acetyl-beta-D-glucosamine pH 6.0, 37°C, mutant D321E isozyme Hex1 Paenibacillus sp. TS12
0.117
-
 4-nitrophenyl N-acetyl-beta-D-glucosamine pH 6.0, 37°C, wild-type isozyme Hex1 Paenibacillus sp. TS12
0.119
-
 4-nitrophenyl N-acetyl-beta-D-glucosamine pH 6.0, 37°C, wild-type isozyme Hex2 Paenibacillus sp. TS12
0.124
-
 4-nitrophenyl N-acetyl-beta-D-galactosamine pH 6.0, 37°C, mutant E322D isozyme Hex1 Paenibacillus sp. TS12
0.127
-
 4-nitrophenyl N-acetyl-beta-D-galactosamine pH 6.0, 37°C, wild-type isozyme Hex1 Paenibacillus sp. TS12
0.159
-
 4-nitrophenyl N-acetyl-beta-D-glucosamine pH 6.0, 37°C, mutant D321N isozyme Hex1 Paenibacillus sp. TS12
0.165
-
 4-nitrophenyl N-acetyl-beta-D-galactosamine pH 6.0, 37°C, mutant E322Q isozyme Hex1 Paenibacillus sp. TS12
0.392
-
 4-nitrophenyl N-acetyl-beta-D-galactosamine pH 6.0, 37°C, mutant D321E isozyme Hex1 Paenibacillus sp. TS12
0.411
-
 4-nitrophenyl N-acetyl-beta-D-galactosamine pH 6.0, 37°C, wild-type isozyme Hex2 Paenibacillus sp. TS12
0.975
-
 4-nitrophenyl N-acetyl-beta-D-galactosamine pH 6.0, 37°C, mutant D321N isozyme Hex1 Paenibacillus sp. TS12

Organism

Organism UniProt Comment Textmining
Paenibacillus sp. TS12
-
 isolated as a GSL-degrading bacterium from soil in Fukuoka Prefecture, Japan, isozymes Hex1 and Hex2
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-type isozymes Hex1 and Hex2, and of Hex1 from Escherichia coli strain BL21(DE3) by affinity chromatography, followed by ion exchange and hydrophobic chromatography Paenibacillus sp. TS12

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4-nitrophenyl N-acetyl-beta-D-galactosamine + H2O
-
 Paenibacillus sp. TS12 4-nitrophenol + N-acetylgalactosamine
-
 ?
4-nitrophenyl N-acetyl-beta-D-glucosamine + H2O preferred substrate Paenibacillus sp. TS12 4-nitrophenol + N-acetylglucosamine
-
 ?
additional information active site and substrate binding structure, and substrate recognition, involving residues Trp352, Trp370, Trp441, Arg170, Asp321, Glu322, Tyl395, Asp397 and Glu443, overview Paenibacillus sp. TS12 ?
-
 ?

Synonyms

Synonyms Comment Organism
beta-HEX
-
 Paenibacillus sp. TS12
Beta-N-acetylhexosaminidase
-
 Paenibacillus sp. TS12
Hex1
-
 Paenibacillus sp. TS12
Hex2
-
 Paenibacillus sp. TS12

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
 assay at Paenibacillus sp. TS12

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
6
-
 assay at Paenibacillus sp. TS12