International Union of Pure and Applied Chemistry

Characterization of a novel rotenoid-b -glucosidase enzyme and its natural substrate from Thai Rosewood*

M.R. Jisnuson Svasti^1,2, Chantragan Srisomsap¹, Rudee Surarit^1,3, Supanna Techasakul^1,4, and James Ketudat-Cairns^1,51. Chulabhorn Research Institute, Bangkok, Thailand
2. Faculty of Science & 3. Faculty of Dentistry, Mahidol University, Bangkok, Thailand
4. Faculty of Science, Kasetsart University, Bangkok, Thailand
5. Instituteof Science, Suranaree University of Technology, Nakorn Ratchasima, Thailand

Abstract: A b -glucosidase enzyme was purified from the seeds of Thai Rosewood (Dalbergia cochinchinensis Pierre) by ammonium sulfate fractionation, preparative isoelectric focusing and Sephadex G-150 chromatography. The electrophoretically homogeneous product had molecular weights of 66,000 in the denatured state and 330,000 in the native state. p-Nitrophenyl-b -D-glucoside (K_m 5.4 mM; k_cat307 sec^-1) and p-NP-b -D-fucoside (K_m 0.54 mM; k_cat 151 sec^-1) were hydrolyzed at the same active site with pH optimum of 5.0. Inhibition was observed with d -gluconolactone, HgCl₂, conduritol B epoxide and Tris. The presumed natural substrate, present in the seed at 3.5% by weight, was purified by silica gel column chromatography and reversed phase TLC, and its structure was determined by NMR. and mass spectroscopy to be a rotenoid b -glucoside. Despite its unusual specificity, sequence analysis indicated that rotenoid b -glucosidase is clearly homologous to other b -glucosidases in its N-terminal sequence. Reversal of the hydrolytic action of Thai Rosewood b -glucosidase by 50% (w/w) D-glucose at 50° C gave disaccharides and trisaccharides in 30% yield, with gentiobiose as the major product. Novel hetero-oligosaccharides could be synthesized from mixtures of D-glucose and D-fucose. Since rotenoids have insecticidal properties, the rotenoid glucoside and its specific b -glucosidase enzyme may play a role in protection against insects.

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