title: Molecular identification of tuliposide B-converting enzyme: a lactone-forming carboxylesterase from the pollen of tulip.
abstract: 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.
authors.affiliation: Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama, 939-0398, Japan.
authors.forename: Taiji
authors.initials: T
authors.lastname: Nomura
authors.affiliation: Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama, 939-0398, Japan.
authors.forename: Tatsunori
authors.initials: T
authors.lastname: Murase
authors.affiliation: Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama, 939-0398, Japan.
authors.forename: Shinjiro
authors.initials: S
authors.lastname: Ogita
authors.affiliation: Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama, 939-0398, Japan.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Kato
mesh_terms: D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D005960:Glucosides / Q000378:metabolism*; D007783:Lactones / Q000378:metabolism*; D008969:Molecular Sequence Data; D011058:Pollen / Q000201:enzymology*; D012333:RNA, Messenger / Q000235:genetics; D031447:Tulipa / Q000201:enzymology*
chemical_list: D005960:Glucosides; D007783:Lactones; D012333:RNA, Messenger; D002265:Carboxylic Ester Hydrolases
keywords: Tulipa gesneriana; lactone-forming carboxylesterase; pollen; tulip; tulipalin; tuliposide; tuliposide-converting enzyme