Literature summary for 5.1.3.11 extracted from

Park, A.R.; Kim, J.S.; Jang, S.W.; Park, Y.G.; Koo, B.S.; Lee, H.C.
Rational modification of substrate binding site by structure-based engineering of a cellobiose 2-epimerase in Caldicellulosiruptor saccharolyticus (2017), Microb. Cell Fact., 16, 224 .

Application

Application	Comment	Organism
synthesis	the enzyme mutant Y114E can be used for production of lactulose for the nutritional industry. Using Y114E, isomerization of lactose to lactulose is optimnized, resulting in 86.9 g/l of lactulose and 4.6 g/l of epilactose for 2 h from 200 g/l of lactose	Caldicellulosiruptor saccharolyticus

Cloned(Commentary)

Cloned (Comment)	Organism
gene Csac_0294, recombinant expressison of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)	Caldicellulosiruptor saccharolyticus

Crystallization (Commentary)

Crystallization (Comment)	Organism
apoenzyme crystal structure comparisons and molecular replacement method using the structure of Ruminococcus albus enzyme (PDB ID 3WKF) as a search model, overview	Caldicellulosiruptor saccharolyticus

Protein Variants

Protein Variants	Comment	Organism
additional information	Y114X mutants are found to lose activity entirely, while the parental enzyme and the Y114E mutant enzyme show increased isomerization activity from lactose to lactulose at 65°C. Isomerase and epimerase activities of wild-type and mutant enzymes, overview	Caldicellulosiruptor saccharolyticus
Y114A	site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C	Caldicellulosiruptor saccharolyticus
Y114C	site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C	Caldicellulosiruptor saccharolyticus
Y114D	site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C	Caldicellulosiruptor saccharolyticus
Y114E	site-directed mutagenesis, the mutation leads to increased release of a byproduct, lactulose, from lactose at 65°C, while its activity is low at 37°C. The Y114E mutation increases isomerization of lactose, while decreasing the epimerization of lactose	Caldicellulosiruptor saccharolyticus
Y114F	site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C	Caldicellulosiruptor saccharolyticus
Y114G	site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C	Caldicellulosiruptor saccharolyticus
Y114H	site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C	Caldicellulosiruptor saccharolyticus
Y114I	site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C	Caldicellulosiruptor saccharolyticus
Y114K	site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C	Caldicellulosiruptor saccharolyticus
Y114L	site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C	Caldicellulosiruptor saccharolyticus
Y114M	site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C	Caldicellulosiruptor saccharolyticus
Y114N	site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C	Caldicellulosiruptor saccharolyticus
Y114P	site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C	Caldicellulosiruptor saccharolyticus
Y114Q	site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C	Caldicellulosiruptor saccharolyticus
Y114R	site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C	Caldicellulosiruptor saccharolyticus
Y114S	site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C	Caldicellulosiruptor saccharolyticus
Y114T	site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C	Caldicellulosiruptor saccharolyticus
Y114V	site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C	Caldicellulosiruptor saccharolyticus
Y114W	site-directed mutagenesis, the mutant shows reduced activity at 37°C compared to wild-type and inactivation at 65°C	Caldicellulosiruptor saccharolyticus

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
beta-1,4-mannobiose	Caldicellulosiruptor saccharolyticus	-	4-O-beta-D-mannopyranosyl-D-glucose	-	r
beta-1,4-mannobiose	Caldicellulosiruptor saccharolyticus ATCC 43494 / DSM 8903 / Tp8T 6331	-	4-O-beta-D-mannopyranosyl-D-glucose	-	r
cellobiose	Caldicellulosiruptor saccharolyticus	-	4-O-beta-D-glucopyranosyl-D-mannose	-	r
cellobiose	Caldicellulosiruptor saccharolyticus ATCC 43494 / DSM 8903 / Tp8T 6331	-	4-O-beta-D-glucopyranosyl-D-mannose	-	r
lactose	Caldicellulosiruptor saccharolyticus	-	epilactose	-	r
lactose	Caldicellulosiruptor saccharolyticus ATCC 43494 / DSM 8903 / Tp8T 6331	-	epilactose	-	r

Organism

Organism	UniProt	Comment	Textmining
Caldicellulosiruptor saccharolyticus	A4XGA6	-	-
Caldicellulosiruptor saccharolyticus ATCC 43494 / DSM 8903 / Tp8T 6331	A4XGA6	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and dialysis	Caldicellulosiruptor saccharolyticus

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
additional information	-	isomerase and epimerase activities of wild-type and mutant enzymes, overview	Caldicellulosiruptor saccharolyticus

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
beta-1,4-mannobiose	-	Caldicellulosiruptor saccharolyticus	4-O-beta-D-mannopyranosyl-D-glucose	-	r
beta-1,4-mannobiose	-	Caldicellulosiruptor saccharolyticus ATCC 43494 / DSM 8903 / Tp8T 6331	4-O-beta-D-mannopyranosyl-D-glucose	-	r
cellobiose	-	Caldicellulosiruptor saccharolyticus	4-O-beta-D-glucopyranosyl-D-mannose	-	r
cellobiose	-	Caldicellulosiruptor saccharolyticus ATCC 43494 / DSM 8903 / Tp8T 6331	4-O-beta-D-glucopyranosyl-D-mannose	-	r
lactose	-	Caldicellulosiruptor saccharolyticus	epilactose	-	r
lactose	-	Caldicellulosiruptor saccharolyticus ATCC 43494 / DSM 8903 / Tp8T 6331	epilactose	-	r

Subunits

Subunits	Comment	Organism
More	three-dimensional structure and substrate binding structure analysis, residues Tyr114 and Asn184 play an important role in binding epilactose, overview. The active site, with its triple histidine center, is located inside the (alpha/alpha)6 barrel structure of the catalytic domain	Caldicellulosiruptor saccharolyticus

Synonyms

Synonyms	Comment	Organism
cellobiose 2-epimerase	-	Caldicellulosiruptor saccharolyticus

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Caldicellulosiruptor saccharolyticus

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Caldicellulosiruptor saccharolyticus

General Information

General Information	Comment	Organism
additional information	three-dimensional structure and substrate binding structure analysis, residues Tyr114 and Asn184 play an important role in binding epilactose, overview. The active site, with its triple histidine center, is located inside the (alpha/alpha)6 barrel structure of the catalytic domain. Two essential conserved histidine residues, His247 and His377, and a third histidine residue located at the bottom of the substrate binding site are required for catalysis. Y114 and N184 in the catalyt domain are located at the interface of the mannose moiety of epilactose in the enzyme-product complex	Caldicellulosiruptor saccharolyticus
physiological function	the enzyme reversibly converts D-glucose residues into D-mannose residues at the reducing end of unmodified beta-1,4-linked oligosaccharides, including beta-1,4-mannobiose, cellobiose, and lactose	Caldicellulosiruptor saccharolyticus