Literature summary for 4.2.2.5 extracted from

Huang, W.; Boju, L.; Tkalec, L.; Su, H.; Yang, H.O.; Gunay, N.S.; Linhardt, R.J.; Kim, Y.S.; Matte, A.; Cygler, M.
Active site of chondroitin AC lyase revealed by the structure of enzyme-oligosaccharide complexes and mutagenesis (2001), Biochemistry, 40, 2359-2372.

Cloned(Commentary)

Cloned (Comment)	Organism
cloning and construction of mutants in Escherichia coli and expression in Flavobacterium heparinum, the Flavobacterium heparinum strains are mutated by conjugation	Pedobacter heparinus

Crystallization (Commentary)

Crystallization (Comment)	Organism
complexes of the wild-type enzyme with dermatan sulfate hexasaccharide, tetrasaccharide, and hyaluronic acid tetrasaccharide, and mutant Y234F enzyme in complex with chondroitin sulfate tetrasaccharide, hanging drop vapour diffusion method, equal volumes, 0.005 ml, of protein and reservoir solution are suspended over 1 ml reservoir solution containing 15% w/v PEG 3350, 0.4 M sodium acetate, 0.1 M HEPES, pH 7.5, at room temperature of about 20°C, 1 day, X-ray diffraction structure determination and analysis at 2.0-2.3 A resolution	Pedobacter heparinus

Crystallization (Comment)

Organism

complexes of the wild-type enzyme with dermatan sulfate hexasaccharide, tetrasaccharide, and hyaluronic acid tetrasaccharide, and mutant Y234F enzyme in complex with chondroitin sulfate tetrasaccharide, hanging drop vapour diffusion method, equal volumes, 0.005 ml, of protein and reservoir solution are suspended over 1 ml reservoir solution containing 15% w/v PEG 3350, 0.4 M sodium acetate, 0.1 M HEPES, pH 7.5, at room temperature of about 20°C, 1 day, X-ray diffraction structure determination and analysis at 2.0-2.3 A resolution

Pedobacter heparinus

Protein Variants

Protein Variants	Comment	Organism
H225A	site-directed mutagenesis, active site residue, inactive mutant	Pedobacter heparinus
R288A	site-directed mutagenesis, active site residue, nearly inactive mutant	Pedobacter heparinus
R292A	site-directed mutagenesis, substrate recognition residue, mutant with residual activity	Pedobacter heparinus
Y234F	site-directed mutagenesis, active site residue, nearly inactive mutant	Pedobacter heparinus

Organism

Organism	UniProt	Comment	Textmining
Pedobacter heparinus	Q59288	gene cslA	-

Purification (Commentary)

Purification (Comment)	Organism
wild-type enzyme, and recombinant mutant enzymes from Flavobacterium heparinum mutant strains	Pedobacter heparinus

Reaction

Reaction	Comment	Organism	Reaction ID
[1,3-beta-D-glucuronic acid-1,4-beta-D-hexosylamine]n = n-1 4-deoxy-beta-D-gluc-4-enuronosyl-1,4-beta-D-hexosylamine + [1,3-beta-D-glucuronic acid-1,4-beta-D-hexosylamine]	acts on chondroitin 4-sulfate and chondroitin 6-sulfate, but less well on hyaluronate, mechanism, active site structure, His225, Tyr234, Arg288, and Glu371 form a catalytic tetrad	Pedobacter heparinus	a

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
0.012	-	purified mutant R292A, substrate dermatan sulfate	Pedobacter heparinus
0.028	-	purified mutant Y234F, substrate dermatan sulfate	Pedobacter heparinus
0.031	-	purified mutant H225A, substrate chondroitin 4-sulfate	Pedobacter heparinus
0.038	-	purified mutant R288A, substrate heparan sulfate	Pedobacter heparinus
0.077	-	purified mutant R292A, substrate heparan sulfate	Pedobacter heparinus
0.12	-	purified mutant Y234F, substrate heparan sulfate	Pedobacter heparinus
0.18	-	purified transconjugated wild-type, substrate heparan sulfate	Pedobacter heparinus
0.2	-	purified transconjugated wild-type, substrate dermatan sulfate	Pedobacter heparinus
0.23	-	purified mutant Y234F, substrate chondroitin 6-sulfate	Pedobacter heparinus
0.3	-	purified mutant Y234F, substrate hyaluronic acid	Pedobacter heparinus
0.45	-	purified mutant R288A, substrate hyaluronic acid	Pedobacter heparinus
0.53	-	purified mutant Y234F, substrate chondroitin 4-sulfate	Pedobacter heparinus
0.55	-	purified mutant R288A, substrate chondroitin 6-sulfate	Pedobacter heparinus
0.94	-	purified mutant R288A, substrate chondroitin 4-sulfate	Pedobacter heparinus
3.63	-	purified mutant R292A, substrate hyaluronic acid	Pedobacter heparinus
9.54	-	purified mutant R292A, substrate chondroitin 6-sulfate	Pedobacter heparinus
21.2	-	purified mutant R292A, substrate chondroitin 4-sulfate	Pedobacter heparinus
62.5	-	purified transconjugated wild-type, substrate chondroitin 6-sulfate	Pedobacter heparinus
94.5	-	purified transconjugated wild-type, substrate hyaluronic acid	Pedobacter heparinus
123.9	-	purified transconjugated wild-type, substrate chondroitin 4-sulfate	Pedobacter heparinus

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
chondroitin sulfate A	i.e. chondroitin 4-sulfate, best substrate, wild-type enzyme and mutants R292A, R288A, H225A and Y234F	Pedobacter heparinus	?	-	?
chondroitin sulfate C	i.e. chondroitin 6-sulfate, wild-type enzyme and mutants R292A, R288A, and Y234F	Pedobacter heparinus	?	-	?
dermatan sulfate	wild-type enzyme and mutants R292A, and Y234F	Pedobacter heparinus	?	-	?
heparan sulfate	wild-type enzyme and mutants R292A, R288A, and Y234F	Pedobacter heparinus	?	-	?
hyaluronic acid	wild-type enzyme and mutants R292A, R288A, and Y234F	Pedobacter heparinus	?	-	?
additional information	Arg292 is involved in recognition of the N-acetyl and sulfate moieties of galactosamine, but does not participate directly in catalysis	Pedobacter heparinus	?	-	?

Synonyms

Synonyms	Comment	Organism
chondroitinase AC	-	Pedobacter heparinus

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8	-	assay at	Pedobacter heparinus