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 (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 | 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 | 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 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 | UniProt | Comment | Textmining |
---|---|---|---|
Paenibacillus sp. TS12 | - |
isolated as a GSL-degrading bacterium from soil in Fukuoka Prefecture, Japan, isozymes Hex1 and Hex2 | - |
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 | 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 | 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 Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Paenibacillus sp. TS12 |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
6 | - |
assay at | Paenibacillus sp. TS12 |