A Novel Lactone-Forming Carboxylesterase: Molecular Identification of a Tuliposide A-Converting Enzyme in Tulip  

Abstract

Tuliposides, the glucose esters of 4-hydroxy-2-methylenebutanoate and 3,4-dihydroxy-2-methylenebutanoate, are major secondary metabolites in tulip (Tulipa gesneriana). Their lactonized aglycons, tulipalins, function as defensive chemicals due to their biological activities. We recently found that tuliposide-converting enzyme (TCE) purified from tulip bulbs catalyzed the conversion of tuliposides to tulipalins, but the possibility of the presence of several TCE isozymes was raised: TCE in tissues other than bulbs is different from bulb TCE. Here, to prove this hypothesis, TCE was purified from petals, which have the second highest TCE activity after bulbs. The purified enzyme, like the bulb enzyme, preferentially accepted tuliposides as substrates, with 6-tuliposide A the best substrate, which allowed naming the enzyme tuliposide A-converting enzyme (TCEA), but specific activity and molecular mass differed between the petal and bulb enzymes. After peptide sequencing, a novel cDNA (TgTCEA) encoding petal TCEA was isolated, and the functional characterization of the recombinant enzyme verified that TgTCEA catalyzes the conversion of 6-tuliposide A to tulipalin A. TgTCEA was transcribed in all tulip tissues but not in bulbs, indicating the presence of a bulb-specific TgTCEA, as suggested by the distinct enzymatic characters between the petal and bulb enzymes. Plastidial localization of TgTCEA enzyme was revealed, which allowed proposing a cytological mechanism of TgTCE-mediated tulipalin formation in the tulip defensive strategy. Site-directed mutagenesis of TgTCEA suggested that the oxyanion hole and catalytic triad characteristic of typical carboxylesterases are essential for the catalytic process of TgTCEA enzyme. To our knowledge, TgTCEA is the first identified member of the lactone-forming carboxylesterases, specifically catalyzing intramolecular transesterification.

Molecular Diversity of Tuliposide A-Converting Enzyme in the Tulip

“…This was the first identified member of the lactone-forming carboxylesterases, specifically catalyzing intramolecular transesterification. 18) The TgTCEA gene was functionally transcribed in the roots, stems, leaves, petals, stamens, and pistils, but no transcription was observed in the bulbs, confirming distinct enzymatic characteristics at least as between bulbs and petals. These facts strongly suggest the possibility that bulb TCEA is encoded by a gene different from the TgTCEA isolated from petals.…”

“…Although all parts of the tulip show TCE activity, 16,18,19) the TCE activity ratios toward 6-tuliposides A and B in the crude extracts differ from tissue to tissue and correlate well with the ratios of 6-tuliposide A and B content in each tissue. 18) Hence the presence of tuliposide B-converting enzyme (TCEB), which preferentially catalyzes the conversion of 6-tuliposide B to tulipalin B, has been suggested ( Fig. 1).…”

“…18) During the course of our characterization of TCEA, we found that enzymatic characters differ between the bulb and petal enzymes with respect to chromatographic behaviors, specific activities, and molecular masses. 18) Most recently, we identified the TgTCEA gene encoding TCEA from tulip petals. This was the first identified member of the lactone-forming carboxylesterases, specifically catalyzing intramolecular transesterification.…”

“…TgTCEA-b shared some features with TgTCEA 6-Tuliposide A is converted to tulipalin A by TCEA. The presence of TCEB, catalyzing the conversion of 6-tuliposide B to tulipalin B, has been suggested, 18) but not yet confirmed. The 1-and 1-anomers are present in 6-tuliposides A and B in tulip plants, and both anomers serve as substrates for TCEs.…”

“…We purified and characterized TCE from tulip bulbs 16) and petals 18) as actively catalyzing the conversion of tuliposides to tulipalins, with 6-tuliposide A the best substrate, and the enzyme was referred to as tuliposide A-converting enzyme (TCEA) (Fig. 1).…”