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

Article abstract of DOI:10.1016/j.abb.2015.07.021

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 k_cat/K_m of 637 mMmM^{-1 -1}. rOs4BGlu13 hydrolyzed helicin best among natural glycosides tested (k_cat/K _m of 74.4 mM^{-1 -1}). Os4BGlu13 was previously designated tuberonic acid glucoside (TAG) β-glucosidase (TAGG), and here the k_cat/K_m of rOsBGlu13 for TAG was 6.68 mM^{-1 -1}, while that for GA₄-GE was 3.63 mM^{-1 -1} and for salicylic acid glucoside (SAG) is 0.88 mM^{-1 -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.