title: Identification of rice Os4BGlu13 as a β-glucosidase which hydrolyzes gibberellin A4 1-O-β-d-glucosyl ester, in addition to tuberonic acid glucoside and salicylic acid derivative glucosides.
abstract: Gibberellin 1-O-β-d-glucose ester hydrolysis activity has been detected in rice seedling extracts, but no enzyme responsible for this activity has ever been purified and identified. Therefore, gibberellin A4 glucosyl ester (GA4-GE) β-d-glucosidase activity was purified from ten-day rice seedling stems and leaves. The family 1 glycoside hydrolase Os4BGlu13 was identified in the final purification fraction. The Os4BGlu13 cDNA was amplified from rice seedlings and expressed as an N-terminal thioredoxin-tagged fusion protein in Escherichia coli. The purified recombinant Os4BGlu13 protein (rOs4BGlu13) had an optimum pH of 4.5, for hydrolysis of p-nitrophenyl β-d-glucopyranoside (pNPGlc), which was the best substrate identified, with a kcat/Km of 637 mM(-1) s(-1). rOs4BGlu13 hydrolyzed helicin best among natural glycosides tested (kcat/Km of 74.4 mM(-1) s(-1)). Os4BGlu13 was previously designated tuberonic acid glucoside (TAG) β-glucosidase (TAGG), and here the kcat/Km of rOsBGlu13 for TAG was 6.68 mM(-1) s(-1), while that for GA4-GE was 3.63 mM(-1) s(-1) and for salicylic acid glucoside (SAG) is 0.88 mM(-1) s(-1). rOs4BGlu13 also hydrolyzed oligosaccharides, with preference for short β-(1 → 3)-linked over β-(1 → 4)-linked glucooligosaccharides. The enzymatic data suggests that Os4BGlu13 may contribute to TAG, SAG, oligosaccharide and GA4-GE hydrolysis in the rice plant, although helicin or a similar compound may be its primary target.
authors.affiliation: The Center for Scientific and Technological Equipment, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand; Center for Biomolecular Structure, Function and Application, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
authors.forename: Yanling
authors.initials: Y
authors.lastname: Hua
authors.affiliation: Center for Biomolecular Structure, Function and Application, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand; School of Biochemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
authors.forename: Watsamon
authors.initials: W
authors.lastname: Ekkhara
authors.affiliation: Department of Chemistry, Faculty of Science, Rajamangala University of Technology, Thanyaburi, Pathun Thani 12110, Thailand.
authors.forename: Sompong
authors.initials: S
authors.lastname: Sansenya
authors.affiliation: Chulabhorn Research Institute, Bangkok 10210, Thailand.
authors.forename: Chantragan
authors.initials: C
authors.lastname: Srisomsap
authors.affiliation: National Center for Genetic Engineering and Biotechnology, Pathum Thani 12120, Thailand.
authors.forename: Sittiruk
authors.initials: S
authors.lastname: Roytrakul
authors.affiliation: Research Faculty of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo 060-8589, Japan.
authors.forename: Wataru
authors.initials: W
authors.lastname: Saburi
authors.affiliation: Research Faculty of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo 060-8589, Japan.
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Takeda
authors.affiliation: Research Faculty of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo 060-8589, Japan.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Matsuura
authors.affiliation: Research Faculty of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo 060-8589, Japan.
authors.forename: Haruhide
authors.initials: H
authors.lastname: Mori
authors.affiliation: Center for Biomolecular Structure, Function and Application, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand; School of Biochemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand; Chulabhorn Research Institute, Bangkok 10210, Thailand. Electronic address: cairns@sut.ac.th.
authors.forename: James R
authors.initials: JR
authors.lastname: Ketudat Cairns
mesh_terms: D002853:Chromatography, Liquid; D003517:Cyclopentanes / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004952:Esters / Q000737:chemistry; D005875:Gibberellins / Q000737:chemistry / Q000378:metabolism*; D005960:Glucosides / Q000378:metabolism*; D006868:Hydrolysis; D013058:Mass Spectrometry; D012275:Oryza / Q000201:enzymology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012459:Salicylates / Q000378:metabolism*; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D003517:Cyclopentanes; D004952:Esters; D005875:Gibberellins; D005960:Glucosides; D011993:Recombinant Fusion Proteins; D012459:Salicylates; C421181:tuberonic acid glucoside; D001617:beta-Glucosidase
keywords: Gibberellin glucosyl ester; Phytohormone conjugates; Recombinant expression; Rice; β-glucosidase