Crystallization (Comment) | Organism |
---|---|
purified wild-type enzyme and mutants E141Q and E162Q, hanging-drop vapor diffusion method, mixing of 4.5-6.5 mg/ml protein with reservoir solution, crystals of E141Q and E162Q mutants are obtained by the micro-seeding method using wild-type crystals as a seed, ligand-bound forms are obtained by the soaking method, 20°C, X-ray diffraction structure determination and analysis at 1.9-2.15 A resolution | Ralstonia sp. |
Protein Variants | Comment | Organism |
---|---|---|
D226A | site-directed mutagenesis, the catalytic site mutant, almost inactive | Ralstonia sp. |
D226N | site-directed mutagenesis, the catalytic site mutant, almost inactive | Ralstonia sp. |
E141A | site-directed mutagenesis, the catalytic site mutant, almost inactive | Ralstonia sp. |
E141D | site-directed mutagenesis, the catalytic site mutant, almost inactive | Ralstonia sp. |
E141N | site-directed mutagenesis, the catalytic site mutant, almost inactive | Ralstonia sp. |
E141Q | site-directed mutagenesis, the catalytic site mutant, almost inactive | Ralstonia sp. |
E162A | site-directed mutagenesis, the mutant shows 70.0% reduced specific activity compared to the wild-type enzyme | Ralstonia sp. |
E162D | site-directed mutagenesis, the mutant shows 153.6% increased specific activity compared to the wild-type enzyme | Ralstonia sp. |
E162N | site-directed mutagenesis, the mutant shows 77.8% reduced specific activity compared to the wild-type enzyme | Ralstonia sp. |
E162Q | site-directed mutagenesis, the mutant shows 83.8% reduced specific activity compared to the wild-type enzyme | Ralstonia sp. |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
Hg+ | - |
Ralstonia sp. |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | steady-state kinetics of the enzymatic reaction toward glycol chitin do not afford accurate values of kcat and Km for the mutant enzymes, except for Glu162 mutants, because of their low activity | Ralstonia sp. |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
additional information | the enzymatic activity is barely affected by metal ions, except for Hg+ | Ralstonia sp. |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
chitin + H2O | Ralstonia sp. | - |
? | - |
? | |
chitin + H2O | Ralstonia sp. A-471 | - |
? | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Ralstonia sp. | B7XCV4 | - |
- |
Ralstonia sp. A-471 | B7XCV4 | - |
- |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
16600 | - |
substrate chitotriose, pH 6.0, 40°C, wild-type enzyme | Ralstonia sp. |
31600 | - |
substrate chitotetraose, pH 6.0, 40°C, wild-type enzyme | Ralstonia sp. |
52600 | - |
substrates chitopentaose and chitohexaose, pH 6.0, 40°C, wild-type enzyme | Ralstonia sp. |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
chitin + H2O | - |
Ralstonia sp. | ? | - |
? | |
chitin + H2O | - |
Ralstonia sp. A-471 | ? | - |
? | |
additional information | identification of five substrate-binding subsites in the enzyme based on crystal structures, enzyme binding specificity, NMR spectroscopy analysis: two ligands, chitobiose and a peptide-glycan fragment, N-acetylglucosaminyl-(beta-1,4)-N-acetylmuramyl-L-alanyl-D-isoglutamine are used for NMR titration experiments: The mutant enzymes except Glu162 mutants E162A, E162Q, E162D, and E162N do not hydrolyze the N-acetyl-beta-D-glucosamine oligomers at all | Ralstonia sp. | ? | - |
? | |
additional information | identification of five substrate-binding subsites in the enzyme based on crystal structures, enzyme binding specificity, NMR spectroscopy analysis: two ligands, chitobiose and a peptide-glycan fragment, N-acetylglucosaminyl-(beta-1,4)-N-acetylmuramyl-L-alanyl-D-isoglutamine are used for NMR titration experiments: The mutant enzymes except Glu162 mutants E162A, E162Q, E162D, and E162N do not hydrolyze the N-acetyl-beta-D-glucosamine oligomers at all | Ralstonia sp. A-471 | ? | - |
? | |
N,N',N'',N''',N'''',N'''''-hexaacetylchitohexaose + H2O | - |
Ralstonia sp. | ? | - |
? | |
N,N',N'',N''',N'''',N'''''-hexaacetylchitohexaose + H2O | - |
Ralstonia sp. A-471 | ? | - |
? | |
N,N',N'',N''',N''''-pentaacetylchitopentaose + H2O | - |
Ralstonia sp. | ? | - |
? | |
N,N',N'',N''',N''''-pentaacetylchitopentaose + H2O | - |
Ralstonia sp. A-471 | ? | - |
? | |
N,N',N'',N'''-tetraacetylchitotetraose + H2O | because the products (N-acetyl-beta-D-glucosamine)3 and N-acetyl-beta-D-glucosamine are rich in alpha- and beta-forms, respectively, (N-acetyl-beta-D-glucosamine)4 is supposed to bind to -2, -1, +1, and +2 subsites and -3, -2, -1, and +1 subsites | Ralstonia sp. | N-acetyl-D-glucosamine + N,N'-diacetylchitobiose + N,N',N''-triacetylchitotriose | - |
? | |
N,N',N''-triacetylchitotriose + H2O | the rate of beta-(N-acetyl-beta-D-glucosamine)3 hydrolysis is higher than that of alpha-(N-acetyl-beta-D-glucosamine)3. Because enzyme Ra-ChiC is an inverting enzyme, chitotriose is supposed to bind predominantly to -2, -1, and +1 subsites | Ralstonia sp. | N,N'-diacetylchitobiose + N-acetyl-D-glucosamine | the products (N-acetyl-beta-D-glucosamine)2 and N-acetyl-beta-D-glucosamine are rich in alpha- and beta-forms, respectively | ? | |
N,N',N''-triacetylchitotriose + H2O | the rate of beta-(N-acetyl-beta-D-glucosamine)3 hydrolysis is higher than that of alpha-(N-acetyl-beta-D-glucosamine)3. Because enzyme Ra-ChiC is an inverting enzyme, chitotriose is supposed to bind predominantly to -2, -1, and +1 subsites | Ralstonia sp. A-471 | N,N'-diacetylchitobiose + N-acetyl-D-glucosamine | the products (N-acetyl-beta-D-glucosamine)2 and N-acetyl-beta-D-glucosamine are rich in alpha- and beta-forms, respectively | ? |
Synonyms | Comment | Organism |
---|---|---|
chitinase C | - |
Ralstonia sp. |
Ra-ChiC | - |
Ralstonia sp. |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
40 | - |
assay at | Ralstonia sp. |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
50 | - |
stable up to | Ralstonia sp. |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
6 | - |
assay at | Ralstonia sp. |
General Information | Comment | Organism |
---|---|---|
evolution | the enzyme belongs to the glycosyl hydrolase family GH23, that also includes goose-type (G-type) lysozymes, peptidoglycan lyases, and peptidoglycan lytic transglycosylases. It has a catalytic domain sequence similar to goose-type (G-type) lysozymes, the unique arrangement of the catalytic residues makes a clear contrast to the other GH23 members and also to inverting GH19 chitinases. Enzyme Ra-ChiC produces alpha-anomers by hydrolyzing beta-1,4-glycosidic linkages, indicating that the enzyme is an inverter like GH family 19 chitinases | Ralstonia sp. |
additional information | structure-function analysis, highly conserved Glu141 acts as a catalytic acid, and Asp226 located at the roof of the tunnel activates a water molecule as a catalytic base, Asp226 is critical for catalysis | Ralstonia sp. |