Cloning of beta-primeverosidase from tea leaves, a key enzyme in tea aroma formation.
A beta-primeverosidase from tea (Camellia sinensis) plants is a unique disaccharide-specific glycosidase, which hydrolyzes aroma precursors of beta-primeverosides (6-O-beta-D-xylopyranosyl-beta-D-glucopyranosides) to liberate various aroma compounds, and the enzyme is deeply concerned with the floral aroma formation in oolong tea and black tea during the manufacturing process. The beta-primeverosidase was purified from fresh leaves of a cultivar for green tea (C. sinensis var sinensis cv Yabukita), and its partial amino acid sequences were determined. The beta-primeverosidase cDNA has been isolated from a cDNA library of cv Yabukita using degenerate oligonucleotide primers. The cDNA insert encodes a polypeptide consisting of an N-terminal signal peptide of 28 amino acid residues and a 479-amino acid mature protein. The beta-primeverosidase protein sequence was 50% to 60% identical to beta-glucosidases from various plants and was classified in a family 1 glycosyl hydrolase. The mature form of the beta-primeverosidase expressed in Escherichia coli was able to hydrolyze beta-primeverosides to liberate a primeverose unit and aglycons, but did not act on 2-phenylethyl beta-D-glucopyranoside. These results indicate that the beta-primeverosidase selectively recognizes the beta-primeverosides as substrates and specifically hydrolyzes the beta-glycosidic bond between the disaccharide and the aglycons. The stereochemistry for enzymatic hydrolysis of 2-phenylethyl beta-primeveroside by the beta-primeverosidase was followed by (1)H-nuclear magnetic resonance spectroscopy, revealing that the enzyme hydrolyzes the beta-primeveroside by a retaining mechanism. The roles of the beta-primeverosidase in the defense mechanism in tea plants and the floral aroma formation during tea manufacturing process are also discussed.
Mizutani
Masaharu
M
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan. mizutani@scl.kyoto-u.ac.jp
Nakanishi
Hidemitsu
H
Ema
Jun-ichi
J
Ma
Seung-Jin
SJ
Noguchi
Etsuko
E
Inohara-Ochiai
Misa
M
Fukuchi-Mizutani
Masako
M
Nakao
Masahiro
M
Sakata
Kanzo
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006031:Glycosylation; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009812:Odorants / Q000032:analysis; D010802:Phylogeny; D018515:Plant Leaves / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins; D017422:Sequence Analysis, DNA; D013662:Tea / Q000737:chemistry / Q000201:enzymology / Q000235:genetics*
D018076:DNA, Complementary; D010940:Plant Proteins; D013662:Tea; D006026:Glycoside Hydrolases; C460087:beta-primeverosidase