Literature summary for 3.2.1.191 extracted from

Shin, K.C.; Hong, S.H.; Seo, M.J.; Oh, D.K.
An amino acid at position 512 in beta-glucosidase from Clavibacter michiganensis determines the regioselectivity for hydrolyzing gypenoside XVII (2015), Appl. Microbiol. Biotechnol., 99, 7987-7996 .

Search for more literature for 3.2.1.191

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of wild-type and mutant enzymes in Escherichia coli strain ER2566	Clavibacter michiganensis

Protein Variants

Protein Variants	Comment	Organism
E332A	site-directed mutagenesis, mutation of a potential catalytic residue, the mutant shows decreased activity with ginsenoside Rb1 and decreased activity with gypenoside XVII compared to the wild-type enzyme	Clavibacter michiganensis
E83A	site-directed mutagenesis, mutation of a potential catalytic residue, the mutant shows increased activity with ginsenoside Rb1 and decreased activity with gypenoside XVII compared to the wild-type enzyme	Clavibacter michiganensis
H238A	site-directed mutagenesis, mutation of a potential catalytic residue, the mutant shows decreased activity with ginsenoside Rb1 and decreased activity with gypenoside XVII compared to the wild-type enzyme	Clavibacter michiganensis
L509A	site-directed mutagenesis, mutation of a potential catalytic residue, the mutant shows slightly decreased activity with ginsenoside Rb1 and decreased activity with gypenoside XVII compared to the wild-type enzyme	Clavibacter michiganensis
additional information	the hydrolytic pathways of enzyme mutants E83A, R131A, R199A, H238A, E332A, and L509A convert GypXVII to GypLXXV, showing the same regioselectivity as the wild-type enzyme. But mutant W512A converts GypXVII to GypLXXV and F2 (cf. EC 3.2.1.193), indicating that the variant simultaneously hydrolyzes the inner glucose linked to the C-3 position and the outer glucose linked to the C-20 position in GypXVII. Mutant W512F variant enzyme containing an aromatic side chain converts GypXVII as a substrate to GypLXXV, while mutant W512R containing a positively charged side chain converts GypXVII to F2	Clavibacter michiganensis
R131A	site-directed mutagenesis, mutation of a potential catalytic residue, the mutant shows decreased activity with ginsenoside Rb1 and decreased activity with gypenoside XVII compared to the wild-type enzyme	Clavibacter michiganensis
R199A	site-directed mutagenesis, mutation of a potential catalytic residue, the mutant shows increased activity with ginsenoside Rb1 and decreased activity with gypenoside XVII compared to the wild-type enzyme	Clavibacter michiganensis
W331A	site-directed mutagenesis, mutation of a potential catalytic residue, inactive mutant	Clavibacter michiganensis
W512A	site-directed mutagenesis, mutation of a potential catalytic residue, the mutant shows slightly decreased activity with ginsenoside Rb1 and decreased activity with gypenoside XVII compared to the wild-type enzyme. Mutant W512A converts GypXVII to GypLXXV and F2 (cf. EC 3.2.1.193)	Clavibacter michiganensis
W512D	site-directed mutagenesis, mutation of a potential catalytic residue, the mutant shows strongly decreased activity with ginsenoside Rb1 and no activity with gypenoside XVII compared to the wild-type enzyme	Clavibacter michiganensis
W512E	site-directed mutagenesis, mutation of a potential catalytic residue, the mutant shows decreased activity with ginsenoside Rb1 and no activity with gypenoside XVII compared to the wild-type enzyme	Clavibacter michiganensis
W512F	site-directed mutagenesis, mutation of a potential catalytic residue, the mutant shows slightly decreased activity with ginsenoside Rb1 and increased activity with gypenoside XVII compared to the wild-type enzyme. Mutant W512F variant enzyme containing an aromatic side chain converts GypXVII as a substrate to GypLXXV	Clavibacter michiganensis
W512K	site-directed mutagenesis, mutation of a potential catalytic residue, the mutant shows strongly decreased activity with ginsenoside Rb1 and with gypenoside XVII compared to the wild-type enzyme	Clavibacter michiganensis
W512R	site-directed mutagenesis, mutation of a potential catalytic residue, the mutant shows strongly decreased activity with ginsenoside Rb1 and with gypenoside XVII compared to the wild-type enzyme. mutant W512R containing a positively charged side chain converts GypXVII to F2, cf. EC 3.2.1.193	Clavibacter michiganensis
Y288A	site-directed mutagenesis, mutation of a potential catalytic residue, inactive mutant	Clavibacter michiganensis

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.18	-	ginsenoside Rb1	pH 7.5, 35°C, recombinant wild-type enzyme	Clavibacter michiganensis
0.2	-	ginsenoside Rb1	pH 7.5, 35°C, recombinant mutant W512A	Clavibacter michiganensis
0.21	-	ginsenoside Rb1	pH 7.5, 35°C, recombinant mutant W512R	Clavibacter michiganensis
0.32	-	gypenoside XVII	pH 7.5, 35°C, recombinant mutant W512R	Clavibacter michiganensis
0.43	-	gypenoside XVII	pH 7.5, 35°C, recombinant mutant W512A	Clavibacter michiganensis
0.46	-	ginsenoside Rb1	pH 7.5, 35°C, recombinant mutant W512E	Clavibacter michiganensis
0.62	-	gypenoside XVII	pH 7.5, 35°C, recombinant wild-type enzyme	Clavibacter michiganensis

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
ginsenoside C-Mc1 + H2O	Clavibacter michiganensis	-	ginsenoside C-Mc + D-glucopyranose	-	?
ginsenoside C-O + H2O	Clavibacter michiganensis	-	ginsenoside C-Y + D-glucopyranose	-	?
ginsenoside F2 + H2O	Clavibacter michiganensis	-	ginsenoside C-K + D-glucopyranose	-	?
ginsenoside Rb1 + H2O	Clavibacter michiganensis	-	gypenoside XVII + D-glucopyranose	-	?
ginsenoside Rb1 + H2O	Clavibacter michiganensis	-	gypenoside XVII + ?	-	?
ginsenoside Rb2 + H2O	Clavibacter michiganensis	-	ginsenoside C-O + D-glucopyranose	-	?
ginsenoside Rc + H2O	Clavibacter michiganensis	-	ginsenoside C-Mc1 + D-glucopyranose	-	?
ginsenoside Rd + H2O	Clavibacter michiganensis	-	ginsenoside F2 + D-glucopyranose	-	?
ginsenoside Rg3 + H2O	Clavibacter michiganensis	-	ginsenoside Rh2 + D-glucopyranose	-	?
ginsenoside Rh2 + H2O	Clavibacter michiganensis	-	protopanaxadiol aglycone + D-glucopyranose	-	?
gypenoside LXXV + H2O	Clavibacter michiganensis	-	ginsenoside C-K + D-glucopyranose	-	?
gypenoside XVII + H2O	Clavibacter michiganensis	-	gypenoside LXXV + D-glucose	-	?
additional information	Clavibacter michiganensis	the specific activity of beta-glucosidase from Clavibacter michiganensis for the PPD-type ginsenosides as substrates followed the order Rb2 > Rd > Rc > Rg3 > Rb1 > compound O (C-O) > compound Mc1 (C-Mc1) > GypXVII > F2 > Rh2 > gypenside LXXV (GypLXXV) and the products of which are C-O, F2, C-Mc1, Rh2, GypXVII, compound Y (C-Y), compound Mc (C-Mc), GypLXXV, compound K (C-K), APPD, and C-K, respectively. The specific activity of beta-glucosidase from Clavibacter michiganensis for the PPT-type ginsenosides as substrates follows the order Rf > Rh1 > Rg1 and the products of which are Rh1, APPT, and F1, respectively	?	-	?

Organism

Organism	UniProt	Comment	Textmining
Clavibacter michiganensis	-	-	-
Clavibacter michiganensis DSM 46364	-	-	-

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
0.0136	-	recombinant mutant W512K, pH 7.5, 35°C, substrate gypenoside XVII	Clavibacter michiganensis
0.0196	-	recombinant mutant W512A, pH 7.5, 35°C, substrate gypenoside XVII	Clavibacter michiganensis
0.0204	-	recombinant mutant H238A, pH 7.5, 35°C, substrate gypenoside XVII	Clavibacter michiganensis
0.0273	-	recombinant mutant R131A, pH 7.5, 35°C, substrate gypenoside XVII	Clavibacter michiganensis
0.0326	-	recombinant mutant L509A, pH 7.5, 35°C, substrate gypenoside XVII	Clavibacter michiganensis
0.0333	-	recombinant mutant W512R, pH 7.5, 35°C, substrate gypenoside XVII	Clavibacter michiganensis
0.0335	-	recombinant mutant R199A, pH 7.5, 35°C, substrate gypenoside XVII	Clavibacter michiganensis
0.0348	-	recombinant mutant E332A, pH 7.5, 35°C, substrate gypenoside XVII	Clavibacter michiganensis
0.0354	-	recombinant mutant E83A, pH 7.5, 35°C, substrate gypenoside XVII	Clavibacter michiganensis
0.0421	-	recombinant wild-type enzyme, pH 7.5, 35°C, substrate gypenoside XVII	Clavibacter michiganensis
0.0647	-	recombinant mutant W512F, pH 7.5, 35°C, substrate gypenoside XVII	Clavibacter michiganensis
1.173	-	recombinant mutant W512K, pH 7.5, 35°C, substrate ginsenoside Rb1	Clavibacter michiganensis
1.248	-	recombinant mutant W512D, pH 7.5, 35°C, substrate ginsenoside Rb1	Clavibacter michiganensis
1.474	-	recombinant mutant W512R, pH 7.5, 35°C, substrate ginsenoside Rb1	Clavibacter michiganensis
1.665	-	recombinant mutant H238A, pH 7.5, 35°C, substrate ginsenoside Rb1	Clavibacter michiganensis
1.896	-	recombinant mutant R131A, pH 7.5, 35°C, substrate ginsenoside Rb1	Clavibacter michiganensis
1.998	-	recombinant mutant E332A, pH 7.5, 35°C, substrate ginsenoside Rb1	Clavibacter michiganensis
2	-	recombinant mutant W512E, pH 7.5, 35°C, substrate ginsenoside Rb1	Clavibacter michiganensis
2.109	-	recombinant mutant L509A, pH 7.5, 35°C, substrate ginsenoside Rb1	Clavibacter michiganensis
2.119	-	recombinant mutant W512F, pH 7.5, 35°C, substrate ginsenoside Rb1	Clavibacter michiganensis
2.135	-	recombinant mutant W512A, pH 7.5, 35°C, substrate ginsenoside Rb1	Clavibacter michiganensis
2.189	-	recombinant wild-type enzyme, pH 7.5, 35°C, substrate ginsenoside Rb1	Clavibacter michiganensis
2.22	-	recombinant mutant E83A, pH 7.5, 35°C, substrate ginsenoside Rb1	Clavibacter michiganensis
2.301	-	recombinant mutant R199A, pH 7.5, 35°C, substrate ginsenoside Rb1	Clavibacter michiganensis

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
ginsenoside C-Mc1 + H2O	-	Clavibacter michiganensis	ginsenoside C-Mc + D-glucopyranose	-	?
ginsenoside C-O + H2O	-	Clavibacter michiganensis	ginsenoside C-Y + D-glucopyranose	-	?
ginsenoside F2 + H2O	-	Clavibacter michiganensis	ginsenoside C-K + D-glucopyranose	-	?
ginsenoside Rb1 + H2O	-	Clavibacter michiganensis	gypenoside XVII + D-glucopyranose	-	?
ginsenoside Rb1 + H2O	-	Clavibacter michiganensis	gypenoside XVII + ?	-	?
ginsenoside Rb2 + H2O	-	Clavibacter michiganensis	ginsenoside C-O + D-glucopyranose	-	?
ginsenoside Rc + H2O	-	Clavibacter michiganensis	ginsenoside C-Mc1 + D-glucopyranose	-	?
ginsenoside Rd + H2O	-	Clavibacter michiganensis	ginsenoside F2 + D-glucopyranose	-	?
ginsenoside Rg3 + H2O	-	Clavibacter michiganensis	ginsenoside Rh2 + D-glucopyranose	-	?
ginsenoside Rh2 + H2O	-	Clavibacter michiganensis	protopanaxadiol aglycone + D-glucopyranose	-	?
gypenoside LXXV + H2O	-	Clavibacter michiganensis	ginsenoside C-K + D-glucopyranose	-	?
gypenoside LXXV + H2O	GypLXXV, low activity	Clavibacter michiganensis	ginsenoside C-K + D-glucopyranose	-	?
gypenoside XVII + H2O	-	Clavibacter michiganensis	gypenoside LXXV + D-glucose	-	?
gypenoside XVII + H2O	GypXVI, enzyme mutants W512A and W512K hydrolyze the inner glucose linked to the C-3 position and the outer glucose linked to the C-20 position of GypXVII to produce GypLXXV and F2. W512R hydrolyzes only the outer glucose linked to the C-20 position of GypXVII to produce F2. Mutants W512E and W512D exhibit no activity for GypXVII	Clavibacter michiganensis	gypenoside LXXV + D-glucose	-	?
additional information	the specific activity of beta-glucosidase from Clavibacter michiganensis for the PPD-type ginsenosides as substrates followed the order Rb2 > Rd > Rc > Rg3 > Rb1 > compound O (C-O) > compound Mc1 (C-Mc1) > GypXVII > F2 > Rh2 > gypenside LXXV (GypLXXV) and the products of which are C-O, F2, C-Mc1, Rh2, GypXVII, compound Y (C-Y), compound Mc (C-Mc), GypLXXV, compound K (C-K), APPD, and C-K, respectively. The specific activity of beta-glucosidase from Clavibacter michiganensis for the PPT-type ginsenosides as substrates follows the order Rf > Rh1 > Rg1 and the products of which are Rh1, APPT, and F1, respectively	Clavibacter michiganensis	?	-	?
additional information	recombinant beta-glucosidase from Clavibacter michiganensis specifically hydrolyzes the outer and inner glucose linked to the C-3 position in protopanaxadiol (PPD)-type ginsenosides (EC 3.2.1.191) and the C-6 position in protopanaxatriol (PPT)-type ginsenosides (EC 3.2.1.194) except for the hydrolysis of gypenoside LXXV (GypLXXV). The enzyme converts gypenoside XVII (GypXVII) to GypLXXV by hydrolyzing the inner glucose linked to the C-3 position	Clavibacter michiganensis	?	-	?

Synonyms

Synonyms	Comment	Organism
More	cf. EC 3.2.1.195, EC 3.2.1.193, and EC 3.2.1.194	Clavibacter michiganensis

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
35	-	-	Clavibacter michiganensis

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
9	-	gypenoside XVII	pH 7.5, 35°C, recombinant mutant W512A	Clavibacter michiganensis
9	-	gypenoside XVII	pH 7.5, 35°C, recombinant mutant W512R	Clavibacter michiganensis
22	-	gypenoside XVII	pH 7.5, 35°C, recombinant wild-type enzyme	Clavibacter michiganensis
343	-	ginsenoside Rb1	pH 7.5, 35°C, recombinant mutant W512R	Clavibacter michiganensis
795	-	ginsenoside Rb1	pH 7.5, 35°C, recombinant mutant W512A	Clavibacter michiganensis
1025	-	ginsenoside Rb1	pH 7.5, 35°C, recombinant wild-type enzyme	Clavibacter michiganensis
1151	-	ginsenoside Rb1	pH 7.5, 35°C, recombinant mutant W512E	Clavibacter michiganensis

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	-	Clavibacter michiganensis

General Information

General Information	Comment	Organism
additional information	the amino acid at position 512 in beta-glucosidase from Clavibacter michiganensis determines the regioselectivity for hydrolyzing gypenoside XVII, obtained from the GypXVII-docked homology models of beta-glucosidase. Homology modeling of Clavibacter michiganensis beta-glucosidase based on the crystal structure of beta-D-glucan exohydrolase from Hordeum vulgare (PDB ID 1EX1) as a template	Clavibacter michiganensis

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
20.9	-	gypenoside XVII	pH 7.5, 35°C, recombinant mutant W512A	Clavibacter michiganensis
28.125	-	gypenoside XVII	pH 7.5, 35°C, recombinant mutant W512R	Clavibacter michiganensis
35.5	-	gypenoside XVII	pH 7.5, 35°C, recombinant wild-type enzyme	Clavibacter michiganensis
1633	-	ginsenoside Rb1	pH 7.5, 35°C, recombinant mutant W512R	Clavibacter michiganensis
2502	-	ginsenoside Rb1	pH 7.5, 35°C, recombinant mutant W512E	Clavibacter michiganensis
3975	-	ginsenoside Rb1	pH 7.5, 35°C, recombinant mutant W512A	Clavibacter michiganensis
5694	-	ginsenoside Rb1	pH 7.5, 35°C, recombinant wild-type enzyme	Clavibacter michiganensis