Literature summary for 3.2.1.14 extracted from

Arimori, T.; Kawamoto, N.; Shinya, S.; Okazaki, N.; Nakazawa, M.; Miyatake, K.; Fukamizo, T.; Ueda, M.; Tamada, T.
Crystal structures of the catalytic domain of a novel glycohydrolase family 23 chitinase from Ralstonia sp. A-471 reveals a unique arrangement of the catalytic residues for inverting chitin hydrolysis (2013), J. Biol. Chem., 288, 18696-18706.

Crystallization (Commentary)

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.

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

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

Inhibitors	Comment	Organism	Structure
Hg+	-	Ralstonia sp.

KM Value [mM]

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

Metals/Ions	Comment	Organism	Structure
additional information	the enzymatic activity is barely affected by metal ions, except for Hg+	Ralstonia sp.

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
chitin + H2O	Ralstonia sp.	-	?	-	?
chitin + H2O	Ralstonia sp. A-471	-	?	-	?

Organism

Organism	UniProt	Comment	Textmining
Ralstonia sp.	B7XCV4	-	-
Ralstonia sp. A-471	B7XCV4	-	-

Specific Activity [micromol/min/mg]

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 and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
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

Synonyms	Comment	Organism
chitinase C	-	Ralstonia sp.
Ra-ChiC	-	Ralstonia sp.

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
40	-	assay at	Ralstonia sp.

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
50	-	stable up to	Ralstonia sp.

pH Optimum

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

General Information

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.