title: Requirement of catalytic-triad and related amino acids for the acyltransferase activity of Tanacetum cinerariifolium GDSL lipase/esterase TcGLIP for ester-bond formation in pyrethrin biosynthesis.
abstract: We have recently discovered that a GDSL lipase/esterase (TcGLIP) in Tanacetum cinerariifolium catalyzed acyltransferase activity to form an ester bond in the natural insecticide, pyrethrin. TcGLIP contained Ser40 in Block I, Gly64 in Block II, Asn168 in Block III and Asp318 and His321 in Block V, suggesting underlying hydrolase activity, although little is known about their role in acyltransferase activity. We expressed TcGLIP here in Esherichia coli as a fusion with maltose-binding protein (MBP), part of the fusion being cleaved with a protease to obtain MBP-free TcGLIP. A kinetic analysis revealed that the MBP moiety scarcely influenced the kinetic parameters. The effects on acyltransferase activity of mutations of Gly64, Asn168, Asp318 and His321 were investigated by using MBP-fused TcGLIP. Mutations of these amino acids markedly reduced the acyltransferase activity, suggesting their critical role in the production of pyrethrins.
authors.affiliation: Dainihon Jochugiku Co., Ltd.
authors.forename: Yukio
authors.initials: Y
authors.lastname: Kikuta
authors.forename: Gen
authors.initials: G
authors.lastname: Yamada
authors.forename: Tomonori
authors.initials: T
authors.lastname: Mitsumori
authors.forename: Takayuki
authors.initials: T
authors.lastname: Takeuchi
authors.forename: Koji
authors.initials: K
authors.lastname: Nakayama
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Katsuda
authors.forename: Akikazu
authors.initials: A
authors.lastname: Hatanaka
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Matsuda
mesh_terms: D000217:Acyltransferases / Q000378:metabolism*; D000595:Amino Acid Sequence; D055162:Biocatalysis; D011723:Chrysanthemum cinerariifolium / Q000201:enzymology*; D004951:Esterification; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D058619:Maltose-Binding Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011722:Pyrethrins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D058619:Maltose-Binding Proteins; D011722:Pyrethrins; D011993:Recombinant Fusion Proteins; D000217:Acyltransferases; D008049:Lipase