Identification and characterization of a GDSL lipase‐like protein that catalyzes the ester‐forming reaction for pyrethrin biosynthesis in Tanacetum cinerariifolium– a new target for plant protection

• Published in: The Plant journal : for cell and molecular biology 2012-07, Vol.71 (2), p.183-193
• Main Authors: Kikuta, Yukio, Ueda, Hirokazu, Takahashi, Masafumi, Mitsumori, Tomonori, Yamada, Gen, Sakamori, Koji, Takeda, Kengo, Furutani, Shogo, Nakayama, Koji, Katsuda, Yoshio, Hatanaka, Akikazu, Matsuda, Kazuhiko
• Format: Article
• Language: English
• Subjects: acyltransferase; Acyltransferases - genetics; Acyltransferases - isolation & purification; Acyltransferases - metabolism; Amino Acid Sequence; Amino Acid Substitution; Biological and medical sciences; Biosynthesis; buds; chrysanthemoyl CoA; Chrysanthemum cinerariaefolium; Chrysanthemum cinerariifolium - chemistry; Chrysanthemum cinerariifolium - cytology; Chrysanthemum cinerariifolium - enzymology; Chrysanthemum cinerariifolium - genetics; coenzyme A; complementary DNA; Escherichia coli; Esterases - genetics; Esterases - isolation & purification; Esterases - metabolism; Esters; extracellular space; flowers; Flowers - enzymology; Flowers - genetics; Fundamental and applied biological sciences. Psychology; GDSL lipase; Gene expression; Gene Expression - genetics; green fluorescent protein; insect control; insect pests; Insecticides; Insecticides - analysis; Insecticides - chemistry; Insecticides - metabolism; Kinetics; leaves; Lipase - genetics; Lipase - isolation & purification; Lipase - metabolism; mature plants; Molecular Sequence Data; Mutagenesis, Site-Directed; mutation; Plant biology; Plant Leaves - enzymology; Plant Leaves - genetics; Plant physiology and development; plant protection; Plant Roots - enzymology; Plant Roots - genetics; Plant Stems - enzymology; Plant Stems - genetics; Protein Sorting Signals; Protein Structure, Tertiary; Proteins; pyrethrin; pyrethrins; Pyrethrins - analysis; Pyrethrins - chemistry; Pyrethrins - metabolism; pyrethroyl CoA; Recombinant Fusion Proteins; seedlings; Seedlings - enzymology; Seedlings - genetics; signal peptide; Substrate Specificity; Tanacetum cinerariifolium
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1111/j.1365-313X.2012.04980.x

Although natural insecticides pyrethrins produced by Tanacetum cinerariifolium are used worldwide to control insect pest species, little information is known of their biosynthesis. From the buds of T. cinerariifolium, we have purified a protein that is able to transfer the chrysanthemoyl group from the coenzyme A (CoA) thioester to pyrethrolone to produce pyrethrin I and have isolated cDNAs that encode the enzyme. To our surprise, the active principle was not a member of a known acyltransferase family but a member of the GDSL lipase family. The recombinant enzyme (TcGLIP) was expressed in Escherichia coli and displayed the acyltransferase reaction with high substrate specificity, recognized the absolute configurations of three asymmetric carbons and also showed esterase activity. A S40A mutation in the Block I domain reduced both acyltransferase and esterase activities, which suggested an important role of this serine residue in these two activities. The signal peptide directed the localization of TcGLIP::enhanced green fluorescent protein (EGFP) fusion, as well as EGFP, to the extracellular space. High TcGLIP gene expression was observed in the leaves of mature plants and seedlings as well as in buds and flowers, a finding that was consistent with the pyrethrin I content in these parts. Expression was enhanced in response to wounding, which suggested that the enzyme plays a key role in the defense mechanism of T. cinerariifolium.