Substrate specificity of tuliposide-converting enzyme,a unique non-ester-hydrolyzing carboxylesterase in tulip: Effects of the alcohol moiety of substrate on the enzyme activity

6-Tuliposides A (PosA) and B (PosB) are glucose esters accumulated in tulip (Tulipa gesneriana) as major defensive secondary metabolites. Pos-converting enzymes (TgTCEs), which we discovered previously from tulip, catalyze the conversion reactions of PosA and PosB to antimicrobial tulipalins A (PaA) and B (PaB), respectively. The TgTCEs, belonging to the carboxylesterase family, specifically catalyze intramolecular transesterification, but not hydrolysis. In this report, we synthesized analogues of Pos with various alcohol moieties, and measured the TgTCE activity together with a determination of the kinetic parameters for these analogues with a view to probe the substrate recognition mechanism of the unique non-ester-hydrolyzing TgTCEs. It was found that d-glucose-like structure and number of the hydroxyl group in alcohol moiety are important for substrate recognition by TgTCEs. Among the analogues examined, 1,2-dideoxy analogues of PosA and PosB were found to be recognized by the TgTCEs more specifically than the authentic substrates by lowering K_m values. The present results will provide a basis for designing simple, stable synthetic substrate analogues for crystallographic analysis of TgTCEs.