title: Biosynthesis of t-anethole in anise: characterization of t-anol/isoeugenol synthase and an O-methyltransferase specific for a C7-C8 propenyl side chain.
abstract: The phenylpropene t-anethole imparts the characteristic sweet aroma of anise (Pimpinella anisum, family Apiaceae) seeds and leaves. Here we report that the aerial parts of the anise plant accumulate t-anethole as the plant matures, with the highest levels of t-anethole found in fruits. Although the anise plant is covered with trichomes, t-anethole accumulates inside the leaves and not in the trichomes or the epidermal cell layer. We have obtained anise cDNA encoding t-anol/isoeugenol synthase 1 (AIS1), an NADPH-dependent enzyme that can biosynthesize t-anol and isoeugenol (the latter not found in anise) from coumaryl acetate and coniferyl acetate, respectively. In addition, we have obtained a cDNA encoding S-[methyl-14C]adenosyl-l-methionine:t-anol/isoeugenol O-methyltransferase 1 (AIMT1), an enzyme that can convert t-anol or isoeugenol to t-anethole or methylisoeugenol, respectively, via methylation of the para-OH group. The genes encoding AIS1 and AIMT1 were expressed throughout the plant and their transcript levels were highest in developing fruits. The AIS1 protein is 59% identical to petunia (Petunia hybrida) isoeugenol synthase 1 and displays apparent Km values of 145 microm for coumaryl acetate and 230 microm for coniferyl acetate. AIMT1 prefers isoeugenol to t-anol by a factor of 2, with Km values of 19.3 microm for isoeugenol and 54.5 microm for S-[methyl-14C]adenosyl-l-methionine. The AIMT1 protein sequence is approximately 40% identical to basil (Ocimum basilicum) and Clarkia breweri phenylpropene O-methyltransferases, but unlike these enzymes, which do not show large discrimination between substrates with isomeric propenyl side chains, AIMT1 shows a 10-fold preference for t-anol over chavicol and for isoeugenol over eugenol.
authors.affiliation: Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109-1048, USA.
authors.forename: Takao
authors.initials: T
authors.lastname: Koeduka
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Baiga
authors.forename: Joseph P
authors.initials: JP
authors.lastname: Noel
authors.forename: Eran
authors.initials: E
authors.lastname: Pichersky
mesh_terms: D000081248:Allylbenzene Derivatives; D000840:Anisoles / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D028042:Pimpinella / Q000201:enzymology* / Q000235:genetics; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D018749:RNA, Plant / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D000081248:Allylbenzene Derivatives; D000840:Anisoles; D018076:DNA, Complementary; D010940:Plant Proteins; D018749:RNA, Plant; D008780:Methyltransferases; C477811:isoeugenol-O-methyltransferase; C006578:anethole