Cloning of beta-primeverosidase from tea leaves, a key enzyme in tea aroma formation

Abstract

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.

Figure 1

SDS-PAGE of fractions from the chromatographic steps of the purification of the β-primeverosidase from fresh green tea leaves. SDS-PAGE was performed using 10% (w/v) polyacrylamide slab gel, and the protein was visualized with silver staining. The migration of size markers is shown to the left of the gel. Lane M, M_r marker; lane 1, the buffer extract; lane 2, acetone precipitate; lane 3, 40% (NH₄)₂SO₄ supernatant; lane 4, Butyl-Toyopearl elute; lane 5, CM-Toyopearl elute; lane 6, Mono S elute.

Figure 2

Nucleotide and predicted amino acid sequences of the β-primeverosidase cDNA from a cultivar for green tea (C. sinensis var sinensis cv Yabukita). Peptide sequences determined from the purified protein are underlined. Arrow indicates the position of the N-terminal amino acid sequence of the purified protein and also the possible cleavage site predicted by PSORT analysis. Possible N-glycosylation sites are boxed. The catalytic residues in the sequence motifs conserved in family 1 glycosyl hydrolases are double underlined.

Figure 3

Deglycosylation of the purified β-primeverosidase with glycopeptidase A. The β-primeverosidase purified from tea leaves was mixed in 1% (w/v) Triton X-100 and 50 mm 2-mercaptoethanol, and heated at 100°C for 15 min. The sample was treated with glycopeptidase A in the buffer for 24 h, and was analyzed by 10% (w/v) SDS-PAGE. The protein was visualized by silver staining. The migration of size makers is shown to the left of the gel. Lane 1, β-Primeverosidase purified from tea leaves; lane 2, β-primeverosidase treated with glycopeptidase A.

Figure 4

A phylogenetic tree of the tea leaf β-primeverosidase with family 1 glycosyl hydrolases from various microorganisms and higher plants. The tree was constructed by alignment of the amino acid sequences using the ClustalW program at the GenomeNet (http://www.genome.ad.jp/) and drawn by the rooted neighbor-joining method. Relative branch lengths are approximately proportional to phylogenetic distance. β-Mannosidase from Pyrococcus furiosus (AAC44387), β-glucosidase from P. furiosus (AAC25555), 6-phospho-β-galactosidase from Streptococcus mutans (AAA16450), 6-phospho-β-glucosidase from Bacillus subtilis (AAA22660), myrosinase from Arabidopsis (AAC18869), myrosinase from Sinapis alba (CAA42534), coniferin β-glucosidase from Pinus contorta (AAC69619), β-glucosidase from Hordeum vulgare (AAA87339), furostanol glycoside 26-O-β-glucosidase from Costus speciosus (BAA11831), linamarase from Manihot esculenta (AAB71381), amygdalin hydrolase from Prunus serotina (PSU26025), prunasin hydrolase from Prunus serotina (AAA93032), linamarase from Trifolium repens (CAA40058), non-cyanogenic β-glucosidase from Trifolium repens (CAA40058), dalcochinin β-glucosidase from Dalbergia cochinchinensis (AAF04007), β-glucosidase from Cucurbita pepo (AAG25897), β-glucosidase from Arabidopsis (AAC16094), indican β-glucosidase from Polygonum tinctorium (BAA78708), β-primeverosidase from C. sinensis var sinensis cv Yabukita (AB088027), raucaffricine-O-β-glucosidase from R. serpentina (AAF03675), strictosidine β-glucosidase from C. roseus (AAF28800), cardenolide 16′-O-glucosidase from Digitalis lanata (CAB38854), cytokinin β-glucosidase from Zea mays (CAA52293), dhurrinase from S. bicolor (AAC49177), β-glucosidase from Avena sativa (AAD02839), β-glucosidase from Secale cereale (AAG00614), cytosolic β-glucosidase from Homo sapiens (CAC08178), β-glucosidase from Spodoptera frugiperda (AAC06038), β-glucosidase from Agrobacterium sp. (AAA22085), β-glucosidase from Streptomyces sp. (CAA82733), and gentiobiase from Bacillus circulans (AAA22266).

Figure 5

SDS-PAGE of the recombinant β-primeverosidase expressed in E. coli. The migration of size marker is shown to the left of the gel. Lane M, M_r marker; lane 1, the supernatant of the crude extracts from the E. coli cells with the expression vector pMALc2-ΔPri; lane 2, the precipitate; lane 3, the recombinant MBP-β-primeverosidase fusion protein purified by an amylose-resin column; lane 4, the MBP-β-primeverosidase fusion protein digested with factor Xa; lane 5, the purified recombinant mature β-primeverosidase.

Figure 6

TLC of the hydrolysis products of pNP β-primeveroside by the recombinant β-primeverosidase. pNP β-primeveroside was incubated with either the β-primeverosidase purified from tea leaves or the recombinant protein produced by E. coli, and the hydrolysates were analyzed by TLC. TLC was carried out on silica gel 60 F254 plates using a solvent system of butanol:pyridine:water:acetic acid (6:4:3:1 [v/v]). Glycosides and sugars were detected by heating the plate at 120°C after spraying with 0.2% (w/v) naphthoresorcinol in H₂SO₄:ethanol (1:19 [v/v]). Lanes 1 through 4 were standard. Lane 1, pNP β-primeveroside; lane 2, Glc; lane 3, Xyl; lane 4, primeverose; lane 5, the reaction products by the β-primeverosidase purified from tea leaves; lane 6, the reaction products by the recombinant mature β-primeverosidase. The arrow indicates the position of primeverose.

Figure 7

Time course of hydrolysis of 2-phenylethyl β-primeveroside catalyzed by the tea leaf β-primeverosidase, followed by ¹H-NMR spectroscopy. A, Postulated retaining hydrolysis of 2-phenylethyl β-primeveroside by the tea enzyme. B, Spectra recorded 0, 5, 10, 20, 40, 90, and 180 min after addition of the enzyme. Ha and He indicate the resonances of H-1 of α- and β-d-Glc, respectively. The full-scan ¹H NMR spectrum of the substrate (2-phenylethyl β-primeveroside) is shown at the bottom.

Figure 8

Distribution of the β-primeverosidase in tea shoots. The acetone powders were prepared from first (including bud), second, third, and fourth leaves, and stem of tea shoots, respectively. The crude extract of each acetone powder was prepared with 10 mm citrate buffer (pH 6.0) by the same procedures as that for the purification. A, Immunoblot analysis was performed by anti-β-primeverosidase antibody. The migration of molecular marker is shown in the left of the gel. Lane 1, Crude extract from the acetone power of tea leaves; lane 2, recombinant His-tagged β-primeverosidase expressed in E. coli. B, β-Primeverosidase activity for each sample was measured with pNP β-primeveroside. C, Immunoblot analysis with the antiprimeverosidase antibody was performed. One microgram of protein was loaded on each lane and separated by 10% (w/v) SDS-PAGE.

Figure 9

Proposed reaction scheme of the tea leaf β-primeverosidase. The tea leaf β-primeverosidase catalyzes the hydrolysis of various kinds of disaccharide β-primeverosides to release a primeverose unit and various aroma compounds by a retaining mechanism but does not sequentially hydrolyze β-primeverosides to monosaccharide units.