TgTCEA from petals TgTCEA and
TgTCEA from petals (TgTCEA1) and TgTCEB from pollen grains (TgTCEB1) were used throughout the study. The recombinant enzymes were expressed in , and purified as the His-tag-free forms as described previously., Enzyme reactions were performed at 4 mM substrates under the same conditions as described previously, and the enzyme activity was calculated based on the amount of PaA or PaB formed by the reaction. We first synthesized the simplest methyl ester analogues of PosA and -PosB, and , respectively, and their -acetylcysteamine (NAC) thioester analogues, and , respectively, according to the methods described in . As shown in (entries 3–6), all these analogues were poor substrates for TgTCEs, suggesting that a structure similar to glucose moiety is crucial for substrate recognition by TgTCEs. No formation of hydroxy acids (PaA- and PaB-acids; prepared as described in ), which are the hydrolytic products of substrates, was observed in the reaction mixture. We then looked into simplifying the alcohol moiety of the substrates, by mimicking a glucose structure. As the simplest molecules, PosA analogues with cyclohexylmethyl and tetrahydropyranylmethyl structures in an alcohol unit, and , respectively, and the synonymous analogues for -PosB, and , respectively, were synthesized as described in , and subjected to the enzyme assay. As is the case in methyl esters and NAC thioesters, these simple analogues were not the preferable substrates for either TgTCE (, entries 7–10), suggesting that the existence of hydroxyl group(s) on the ring moiety is requisite for substrate recognition by TgTCEs. Firstly, in order to examine the effect of a hydroxyl group at the C-1 position of glucose, α-1--Me analogues of PosA and -PosB, and , and their β-1--Me analogues, and , were synthesized as described in , and subjected to the enzyme assay. As shown in (entries 11, 12, 14, and 15), protection of the C-1 hydroxyl group enabled TgTCEA to better recognize A-type substrates, whereas the opposite effect was observed for TgTCEB, indicating that TgTCEA and TgTCEB recognize the hydroxyl group at the C-1 position of glucose in a distinct manner. We next designed substrate analogues by sequentially adding hydroxyl groups to the tetrahydropyranylmethyl moiety; starting from the analogues having the C-4 hydroxyl group, we synthesized 1,2,3-trideoxy analogues of PosA and -PosB, and (), respectively, and then synthesized 1,2-dideoxy analogues, and (), respectively. With the results obtained by the enzyme assay (, entries 17–20), we identified 1,2-dideoxy analogues as acceptable substrates for both TgTCEs, especially for TgTCEA, while 1,2,3-trideoxy analogues were not recognized as good substrates by either TgTCE. Of the 16 synthetic analogues, compounds , , and ( and for each) served as substantial substrates. We thus synthesized chiral (()-form) acyl unit corresponding to natural PosB as described in , and the THZ2 was condensed with α- or β-1--Me, or with 1,2-dideoxy type alcohol units, to produce chiral PosB analogues (, , and ) as described in . Using these analogues, we measured the enzyme activity (, entries 13, 16, and 21) and found that stereochemistry at acyl side chain does not greatly affect the enzyme activity except for TgTCEB activity toward , which was 3-fold higher than that toward . We next determined the kinetic parameters of TgTCEA and TgTCEB with A-type analogues (, , and ) and chiral B-type analogues (, , and ), and compared with those for authentic substrates, PosA and PosB. The kinetic parameters of TgTCEA and TgTCEB are listed in , , respectively (see , respectively, for Michaelis-Menten plots). Catalytic efficiency (/) of TgTCEA did not differ greatly between the authentic substrate PosA and its analogues (). The ratio of / values for PosA/PosB was 27.7, and the ratio increased approximately three times by changing their alcohol moiety; the values for /, /, and / were 87.0, 76.4, and 80.2, respectively. These results suggest that the deletion or protection of the C-1 hydroxyl group of substrate improves TgTCEA discrimination between A- and B-type substrates. This is mainly due to the decrease in the values for the B-type analogues to approximately one-tenth to one-fourth of PosB while those for A-type substrates were comparable. On the other hand, the opposite phenomenon was observed for TgTCEB (). The ratio for PosB/PosA of TgTCEB was 238, and the ratio decreased by changing their alcohol moiety to α- or β-1--Me forms; the values for / and / were 59.8 and 62.8, respectively. These changes are due to the remarkable decrease in the values, without affecting the values, for B-type analogues by the protection of the C-1 hydroxyl group. Interestingly, the value for / of TgTCEB was 186, which was comparable with that for PosB/PosA; value for was approximately one-third of that for PosB, while the value for PosB was five times higher than that for . Very recently, during the course of studies on an action of TgTCEs against naturally-occurring Pos derivatives, we found that the presence of a 1-acyl group increases the affinity of the enzymes for substrates, rather than interferes with substrate binding; the values of TgTCEA and TgTCEB for PosD and PosF, which are naturally-occurring 1,6-diacyl type analogues of PosA and PosB, respectively, were remarkably lower than those for PosA and PosB, respectively. Due to mutarotation of the glucose moiety, 1α- and 1β-anomers exist for PosA and PosB, and both anomers serve as substrates. Therefore, preferences of TgTCEA and TgTCEB for PosA and PosB, respectively, had been considered to be primarily dependent on the structure of the 6-acyl groups, but not on the configuration of the C-1 position. However, given the results obtained here, not only the structure of the 6-acyl group, but also the structure and the configuration of the C-1 position of substrate appear to be involved in the preferences of TgTCEA and TgTCEB for PosA and PosB, respectively.