Literature summary for 3.8.1.5 extracted from

Okai, M.; Ohtsuka, J.; Imai, L.F.; Mase, T.; Moriuchi, R.; Tsuda, M.; Nagata, K.; Nagata, Y.; Tanokura, M.
Crystal structure and site-directed mutagenesis analyses of haloalkane dehalogenase LinB from Sphingobium sp. strain MI1205 (2013), J. Bacteriol., 195, 2642-2651.

Cloned(Commentary)

Cloned (Comment)	Organism
gene linB, DNA and amino acid sequence determination and analysis, genotyping and sequence comparisons	Sphingobium indicum
gene linB, DNA and amino acid sequence determination and analysis, genotyping and sequence comparisons, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)	Sphingobium sp.

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant wild-type LinBMI, sitting drop vapor diffusion method, mixing of equal volumes of 0.001 ml of the protein solution and reservoir solution, containing 100mM Tris-HCl, pH 8.8-9.0, 200 mM CaCl2, and 17-19% w/v PEG 6000 for untagged enzyme and 100 mM Tris-HCl, pH 8.5, 200 mM MgCl2. and 20% w/v PEG 4000 for His-tagged enzyme, and equilibration against 0.3 ml of reservoir solution at 4°C or 20°C, X-ray diffraction structure determination and analysis, structure modeling	Sphingobium sp.

Crystallization (Comment)

Organism

purified recombinant wild-type LinBMI, sitting drop vapor diffusion method, mixing of equal volumes of 0.001 ml of the protein solution and reservoir solution, containing 100mM Tris-HCl, pH 8.8-9.0, 200 mM CaCl2, and 17-19% w/v PEG 6000 for untagged enzyme and 100 mM Tris-HCl, pH 8.5, 200 mM MgCl2. and 20% w/v PEG 4000 for His-tagged enzyme, and equilibration against 0.3 ml of reservoir solution at 4°C or 20°C, X-ray diffraction structure determination and analysis, structure modeling

Sphingobium sp.

Protein Variants

Protein Variants	Comment	Organism
H247A	site-directed mutagenesis	Sphingobium sp.
I253M	site-directed mutagenesis	Sphingobium sp.
L138I	site-directed mutagenesis	Sphingobium sp.
additional information	mutations are constructed corresponding to the residues in LinBUT from Sphingobium japonicum strain UT26	Sphingobium sp.
T135A	site-directed mutagenesis	Sphingobium sp.
T81A	site-directed mutagenesis	Sphingobium sp.
V112A	site-directed mutagenesis	Sphingobium sp.
V134I	site-directed mutagenesis	Sphingobium sp.

Organism

Organism	UniProt	Comment	Textmining
Sphingobium indicum	-	-	-
Sphingobium indicum UT26	-	-	-
Sphingobium sp.	A4PEU6	-	-
Sphingobium sp. MI1205	A4PEU6	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by metal affinity chromatography	Sphingobium sp.

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
2,3,4,5,6-pentachlorocyclohexanol + H2O	-	Sphingobium indicum	2,3,5,6-tetrachlorocyclohexane-1,4-diol + chloride	-	?
2,3,4,5,6-pentachlorocyclohexanol + H2O	-	Sphingobium sp.	2,3,5,6-tetrachlorocyclohexane-1,4-diol + chloride	-	?
2,3,4,5,6-pentachlorocyclohexanol + H2O	-	Sphingobium sp. MI1205	2,3,5,6-tetrachlorocyclohexane-1,4-diol + chloride	-	?
2,3,4,5,6-pentachlorocyclohexanol + H2O	-	Sphingobium indicum UT26	2,3,5,6-tetrachlorocyclohexane-1,4-diol + chloride	-	?
beta-hexachlorocyclohexane + H2O	-	Sphingobium indicum	2,3,4,5,6-pentachlorocyclohexanol + chloride	-	?
beta-hexachlorocyclohexane + H2O	-	Sphingobium sp.	2,3,4,5,6-pentachlorocyclohexanol + chloride	-	?
beta-hexachlorocyclohexane + H2O	-	Sphingobium sp. MI1205	2,3,4,5,6-pentachlorocyclohexanol + chloride	-	?
beta-hexachlorocyclohexane + H2O	-	Sphingobium indicum UT26	2,3,4,5,6-pentachlorocyclohexanol + chloride	-	?

Synonyms

Synonyms	Comment	Organism
LinB	-	Sphingobium indicum
LinB	-	Sphingobium sp.
LinBMI	-	Sphingobium sp.
LinBUT	-	Sphingobium indicum

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	assay at	Sphingobium sp.

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Sphingobium sp.

General Information

General Information	Comment	Organism
evolution	the enzymes LinBUT and LinBMI, i.e. LinB from Sphingobium japonicum UT26 and Sphingobium sp. MI1205, respectively, catalyze the hydrolytic dechlorination of beta-hexachlorocyclohexane, mutational analysis and sequence comparisons, overview	Sphingobium indicum
evolution	the enzymes LinBUT and LinBMI, i.e. LinB from Sphingobium japonicum UT26 and Sphingobium sp. MI1205, respectively, catalyze the hydrolytic dechlorination of beta-hexachlorocyclohexane, mutational analysis and sequence comparisons, overview	Sphingobium sp.
additional information	enzyme ligand docking and molecular dynamics simulations, structure comparisons of wild-type and mutant enzymes, effects of different residues located near the active site on the specificity constants, overview	Sphingobium sp.

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.0036	-	2,3,4,5,6-pentachlorocyclohexanol	pH 7.5, 30°C, recombinant wild-type enzyme	Sphingobium indicum
0.01	-	beta-hexachlorocyclohexane	pH 7.5, 30°C, recombinant mutant V112A	Sphingobium sp.
0.027	-	2,3,4,5,6-pentachlorocyclohexanol	pH 7.5, 30°C, recombinant mutant V134I/H247A	Sphingobium sp.
0.0271	-	beta-hexachlorocyclohexane	pH 7.5, 30°C, recombinant wild-type enzyme	Sphingobium indicum
0.07	-	beta-hexachlorocyclohexane	pH 7.5, 30°C, recombinant mutant T81A	Sphingobium sp.
0.08	-	beta-hexachlorocyclohexane	pH 7.5, 30°C, recombinant mutant T135A	Sphingobium sp.
0.08	-	2,3,4,5,6-pentachlorocyclohexanol	pH 7.5, 30°C, recombinant mutant V134L	Sphingobium sp.
0.104	-	beta-hexachlorocyclohexane	pH 7.5, 30°C, recombinant mutant V134I/H247A	Sphingobium sp.
0.124	-	beta-hexachlorocyclohexane	pH 7.5, 30°C, recombinant mutant V134L	Sphingobium sp.
0.13	-	beta-hexachlorocyclohexane	pH 7.5, 30°C, recombinant mutant L138I	Sphingobium sp.
0.14	-	2,3,4,5,6-pentachlorocyclohexanol	pH 7.5, 30°C, recombinant mutant I253M	Sphingobium sp.
0.19	0.205	beta-hexachlorocyclohexane	pH 7.5, 30°C, recombinant wild-type enzyme	Sphingobium sp.
0.21	-	beta-hexachlorocyclohexane	pH 7.5, 30°C, recombinant mutant H247A	Sphingobium sp.
0.21	-	beta-hexachlorocyclohexane	pH 7.5, 30°C, recombinant mutant I253M	Sphingobium sp.
0.22	-	2,3,4,5,6-pentachlorocyclohexanol	pH 7.5, 30°C, recombinant mutant L138I	Sphingobium sp.
0.23	-	2,3,4,5,6-pentachlorocyclohexanol	pH 7.5, 30°C, recombinant mutant V112A	Sphingobium sp.
0.24	-	2,3,4,5,6-pentachlorocyclohexanol	pH 7.5, 30°C, recombinant mutant H247A	Sphingobium sp.
0.42	-	2,3,4,5,6-pentachlorocyclohexanol	pH 7.5, 30°C, recombinant mutant T135A	Sphingobium sp.
0.72	1	2,3,4,5,6-pentachlorocyclohexanol	pH 7.5, 30°C, recombinant wild-type enzyme	Sphingobium sp.
0.95	-	2,3,4,5,6-pentachlorocyclohexanol	pH 7.5, 30°C, recombinant mutant T81A	Sphingobium sp.