Literature summary for 3.2.1.149 extracted from

Saino, H.; Shimizu, T.; Hiratake, J.; Nakatsu, T.; Kato, H.; Sakata, K.; Mizutani, M.
Crystal structures of beta-primeverosidase in complex with disaccharide amidine inhibitors (2014), J. Biol. Chem., 289, 16826-16834.

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Cloned(Commentary)

Cloned (Comment)	Organism
sequence comparison, the enzyme including the N-terminal signal sequence is cloned and expressed in a baculovirus-insect cell system	Camellia sinensis

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant alpha-mannosidase-treated enzyme, in complex with disaccharide amidine inhibitors, sitting drop vapor diffusion method, mixing of 0.0015 ml of 10 mg/ml protein in 10mM sodium acetate, with 0.0015 ml of reservoir solution containing 0.01% Triton X-100, 21% PEG 4000, 0.3 M ammonium acetate, and 0.1 M sodium citrate, pH 5.5, 20°C, X-ray diffraction structure determination and analysis at 1.8-1.9 A resolution, molecular replacement	Camellia sinensis

Crystallization (Comment)

Organism

purified recombinant alpha-mannosidase-treated enzyme, in complex with disaccharide amidine inhibitors, sitting drop vapor diffusion method, mixing of 0.0015 ml of 10 mg/ml protein in 10mM sodium acetate, with 0.0015 ml of reservoir solution containing 0.01% Triton X-100, 21% PEG 4000, 0.3 M ammonium acetate, and 0.1 M sodium citrate, pH 5.5, 20°C, X-ray diffraction structure determination and analysis at 1.8-1.9 A resolution, molecular replacement

Camellia sinensis

Inhibitors

Inhibitors	Comment	Organism	Structure
2-phenyl-N-(6-O-beta-D-xylopyranosyl-beta-D-glucopyranosyl)ethylamidine	enzyme binding structure, overview	Camellia sinensis
4-(N-benzylsuccinimide-3-sulfanyl)-N-(6-O-beta-D-xylopyranosyl-beta-D-glucopyranosyl)butylamidine	enzyme binding structure, overview	Camellia sinensis

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
a 6-O-(beta-D-xylopyranosyl)-beta-D-glucopyranoside + H2O	Camellia sinensis	-	6-O-(beta-D-xylopyranosyl)-beta-D-glucopyranose + an alcohol	-	?

Organism

Organism	UniProt	Comment	Textmining
Camellia sinensis	Q7X9A9	-	-

Posttranslational Modification

Posttranslational Modification	Comment	Organism
glycoprotein	deglycosylation by alpha-mannosidase	Camellia sinensis

Purification (Commentary)

Purification (Comment)	Organism
recombinant enzyme from insect cells by cation exchange chromatography and ConA affinity chromatography with N-beta-primeverosylamidine adsorbent	Camellia sinensis

Source Tissue

Source Tissue	Comment	Organism	Textmining
leaf	-	Camellia sinensis	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
2-phenylethyl beta-D-glucopyranoside + H2O	low activity	Camellia sinensis	2-phenylethanol + beta-D-glucopyranose	-	?
2-phenylethyl beta-primeveroside + H2O	-	Camellia sinensis	2-phenylethanol + beta-primeverose	-	?
a 6-O-(beta-D-xylopyranosyl)-beta-D-glucopyranoside + H2O	-	Camellia sinensis	6-O-(beta-D-xylopyranosyl)-beta-D-glucopyranose + an alcohol	-	?
a 6-O-(beta-D-xylopyranosyl)-beta-D-glucopyranoside + H2O	the enzyme hydrolyzes beta-primeveroside at the beta-glycosidic bond of primeverose to aglycone, and releases aromatic alcoholic volatiles of aglycones	Camellia sinensis	6-O-(beta-D-xylopyranosyl)-beta-D-glucopyranose + an alcohol	-	?
additional information	the enzyme only accepts primeverose as the glycone substrate, but broadly accepts various aglycones, including 2-phenylethanol, benzyl alcohol, linalool, and geraniol. Compared with aglycone-specific beta-glucosidases of the glycoside hydrolase family 1, beta-primeverosidase lacks the Trp crucial for aglycone recognition, and the resultant large cavity accepts aglycone and 6-O-beta-D-xylopyranosyl together. The enzyme barely hydrolyzes other beta-glycosides, in which the beta-1,6-Xyl group is absent or substituted by other sugars, such as monosaccharide glycosides (beta-D-glucopyranoside and beta-D-xylopyranoside, etc.) or other disaccharide glycosides, beta-vicianosides (6-O-alpha-L-arabinopyranosyl-beta-D-glucopyranoside), beta-gentiobiosides (6-O-beta-D-glucopyranosyl-beta-D-glucopyranoside), and beta-acuminosides (6-O-beta-D-apiofuranosyl-beta-D-glucopyranoside)	Camellia sinensis	?	-	?

Ki Value [mM]

Ki Value [mM]	Ki Value maximum [mM]	Inhibitor	Comment	Organism	Structure
0.026	-	4-(N-benzylsuccinimide-3-sulfanyl)-N-(6-O-beta-D-xylopyranosyl-beta-D-glucopyranosyl)butylamidine	recombinant His-tagged enzyme, pH and temperature not specified in the publication	Camellia sinensis

General Information

General Information	Comment	Organism
additional information	three-dimensional structures of the enzyme in apo and complex forms with N-beta-primeverosylamidines, overview. Three subsites in the active site: subsite -2 specific for 6-O-beta-D-xylopyranosyl, subsite -1 well conserved among beta-glucosidases and specific for beta-D-glucopyranosyl, and wide subsite +1 for hydrophobic aglycone. Glu470, Ser473, and Gln477 act as the specific hydrogen bond donors for 6-O-beta-D-xylopyranosyl in subsite +2. On the other hand, subsite +1 is a large hydrophobic cavity that accommodates various aromatic aglycones. Tight binding of the disaccharide in the deep active site, overview	Camellia sinensis
physiological function	beta-primeverosidase is a disaccharide-specific beta-glycosidase in tea leaves. This enzyme is involved in aroma formation during the manufacturing process of oolong tea and black tea. The enzyme hydrolyzes beta-primeveroside at the beta-glycosidic bond of primeverose to aglycone, and releases aromatic alcoholic volatiles of aglycones	Camellia sinensis