br Conclusion br Experimental NMR spectra were recorded on
Experimental NMR spectra were recorded on a Jeol spectrometer at either 270 or 400 MHz for 1H spectra and either 68 or 100 MHz for 13C spectra. All chemical shifts are quoted in ppm relative to tetramethylsilane. In the assignment of signals the abbreviation DHN is used for 2,3-dihydroxynaphthalene and CHA for cyclohexylamine. Optical rotations were measured on an Optical Activity AA10 polarimeter. High resolution mass spectra (HRMS) were obtained from the EPSRC Mass Spectrometry Facility (Swansea). Flash chromatography was performed using Fluorochem Ltd silica gel (60 Å). Mixed solvents are recorded as volumetric ratios. EtOH is methylated spirit. Water is deionised water. HPLC was performed using an Agilent 1260 modular HPLC instrument fitted with a multiple wavelength detector and a Phenomenex Kinetex 2.6 μ C18, 150 × 2.1 mm column. Data was acquired at 280 nm with a flow rate of 0.4 mL per minute at ambient temperature. Mobile phase A was water containing 0.1% v/v formic Metabolism Compound Library australia and mobile phase B was acetonitrile containing 0.1% v/v formic acid. Substrates were prepared in a mixture 95% A/ 5% B. The gradient used was: 0–10 mins 95% A/ 5% B; 10-13.5 mins 5% A/ 95% B; 13.5-18 mins 95% A/ 5% B. The HPLC traces of key substrates are shown in the supplementary information.
6-Tuliposides (Pos), the major secondary metabolites in tulip (), are glucose esters of 4-hydroxy-2-methylenebutanoate and (3)-3,4-dihydroxy-2-methylenebutanoate; the former and the latter are referred to as PosA and PosB, respectively ()., , PosA and PosB serve as precursors of the antimicrobial α-methylene-γ-butyrolactones, tulipalins A (PaA) and B (PaB), respectively, which are formed from the hydroxyl acids at the C-6 position of -glucose ()., , We previously discovered a unique Pos-converting enzymes (tuliposide-converting enzymes, TgTCEs) that specifically catalyze the conversion reactions of Pos to Pa ()., , , , , , , , Two distinct types of TgTCE, PosA-converting enzyme (TgTCEA) and PosB-converting enzyme (TgTCEB), are present in tulip tissues, and several isozymes with distinct expression profiles in tulip tissues have been identified for each of TgTCEA and TgTCEB (): TgTCEA from bulbs, and petals, and TgTCEB from pollen grains, roots, and leaves. Both types of TgTCE belong to the plant class I carboxylesterase family in the α/β-hydrolase fold superfamily. Canonical carboxylesterase catalyzes the hydrolysis of a carboxylic ester to form a carboxylic acid and an alcohol, but TgTCEs catalyze only intramolecular transesterification of Pos to form Pa and -glucose in a stoichiometric manner, and never catalyze hydrolysis of Pos to form hydroxy acids (). Hence, TgTCEs were identified as unique “non-ester-hydrolyzing carboxylesterases” and classified as lyases (EC 220.127.116.11 for TgTCEA and EC 18.104.22.168 for TgTCEB), but not as hydrolases. The enzyme reaction by TgTCEs begins with a nucleophilic attack by the catalytic Ser, whose hydroxyl group is activated by the charge relay of the catalytic triad, on the carbonyl carbon of Pos. This is followed by the formation of a tetrahedral intermediate, which is stabilized by the oxyanion hole structure formed by the two Gly residues of the HGG motif. Then, following the elimination of glucose, the acyl-enzyme complex is formed, and an intramolecular nucleophilic attack by a terminal hydroxyl group of Pos, but not by water, occurs, and this nucleophilic attack results in the formation of the five-membered ring structure of Pa. Although we proposed this reaction mechanism based on that of the canonical ester-hydrolyzing carboxylesterase and the site-directed mutagenesis analysis of the enzyme, it has not yet been verified experimentally by the crystallographic analysis. Moreover, there has so far been no information regarding the requisite substrate structures to be recognized by TgTCEs. This prompted us to examine the structure-activity relationship using structural analogues of Pos. Considering that natural Pos are chemically labile under neutral to basic conditions, such information is also useful to design more simple, stable substrate mimics that can be applied to co-crystallization experiments with TgTCEs, which lead to the verification of the reaction mechanism, including the formation of tetrahedral intermediate and the intramolecular nucleophilic attack. We hereby focused on the structural requirements of the alcohol moiety of Pos for recognition by TgTCEs. We synthesized many analogues of PosA and PosB by combinations of acyl units (for PosA, racemic ()-PosB, and PosB) with various alcohol units (), and assessed their effects on the enzyme activities of TgTCEA and TgTCEB. Moreover, stabilities of the synthetic analogues in aqueous solution were examined and compared with those of natural substrates, PosA and PosB.