Molecular identification of tuliposide B-converting enzyme: a lactone-forming carboxylesterase from the pollen of tulip.
Molecular identification of tuliposide B-converting enzyme: a lactone-forming carboxylesterase from the pollen of tulip.
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DOI：
10.1111/tpj.12883
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发表时间：
2015-07
期刊：
影响因子：
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通讯作者：
Taiji Nomura;T. Murase;S. Ogita;Y. Kato
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文献类型：
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作者：
Taiji Nomura;T. Murase;S. Ogita;Y. Kato
6-郁金香苷 A (PosA) 和 B (PosB) 是郁金香 (Tulipa gesneriana) 中的主要次级代谢产物，可分别通过酶促转化为抗菌内酯化苷元郁金香素 A (PaA) 和 B (PaB)。我们最近发现 PosA 转换酶 (TCEA) 是第一个报道的内酯形成羧酸酯酶成员。在此，我们描述了另一种形成内酯的羧酸酯酶 PosB 转换酶 (TCEB) 的鉴定，它优先与 PosB 反应生成 PaB。该酶是从郁金香花粉中分离出来的，具有很高的 PosB 转换活性。纯化的 TCEB 对 PosB 的活性高于 PosA，这与 TCEA 的活性相反。从郁金香花粉中分离出编码 TCEB 的新 cDNA (TgTCEB1)。 TgTCEB1属于羧酸酯酶家族并且与TgTCEA多肽大约50%相同。重组酶的功能表征证实，TgTCEB1 催化 PosB 向 PaB 的转化，其活性与天然 TCEB 相当。对植物各部分的 RT-qPCR 分析表明，TgTCEB1 转录物几乎只限于花粉。此外，使用抗TgTCEB1多克隆抗体对花药横截面进行免疫染色，证实TgTCEB1在花粉粒中特异性表达，但在花药细胞中不表达。 TgTCEB1 的 N 端转运肽被证明具有质体靶向信号的功能。综上所述，这些结果表明成熟的 TgTCEB1 特异性定位于花粉粒的质体中。有趣的是，TgTCEB1 的底物 PosB 积聚在花粉表面，而不是花粉粒的细胞内空间。
6-Tuliposides A (PosA) and B (PosB), which are the major secondary metabolites in tulip (Tulipa gesneriana), are enzymatically converted to the antimicrobial lactonized aglycons, tulipalins A (PaA) and B (PaB), respectively. We recently identified a PosA-converting enzyme (TCEA) as the first reported member of the lactone-forming carboxylesterases. Herein, we describe the identification of another lactone-forming carboxylesterase, PosB-converting enzyme (TCEB), which preferentially reacts with PosB to give PaB. This enzyme was isolated from tulip pollen, which showed high PosB-converting activity. Purified TCEB exhibited greater activity towards PosB than PosA, which was contrary to that of the TCEA. Novel cDNA (TgTCEB1) encoding the TCEB was isolated from tulip pollen. TgTCEB1 belonged to the carboxylesterase family and was approximately 50% identical to the TgTCEA polypeptides. Functional characterization of the recombinant enzyme verified that TgTCEB1 catalyzed the conversion of PosB to PaB with an activity comparable with the native TCEB. RT-qPCR analysis of each part of plant revealed that TgTCEB1 transcripts were limited almost exclusively to the pollen. Furthermore, the immunostaining of the anther cross-section using anti-TgTCEB1 polyclonal antibody verified that TgTCEB1 was specifically expressed in the pollen grains, but not in the anther cells. N-terminal transit peptide of TgTCEB1 was shown to function as plastid-targeted signal. Taken together, these results indicate that mature TgTCEB1 is specifically localized in plastids of pollen grains. Interestingly, PosB, the substrate of TgTCEB1, accumulated on the pollen surface, but not in the intracellular spaces of pollen grains.