title: An unusual plant triterpene synthase with predominant α-amyrin-producing activity identified by characterizing oxidosqualene cyclases from Malus × domestica.
abstract: The pentacyclic triterpenes, in particular ursolic acid and oleanolic acid and their derivatives, exist abundantly in the plant kingdom, where they are well known for their anti-inflammatory, antitumour and antimicrobial properties. α-Amyrin and β-amyrin are the precursors of ursolic and oleanolic acids, respectively, formed by concerted cyclization of squalene epoxide by a complex synthase reaction. We identified three full-length expressed sequence tag sequences in cDNA libraries constructed from apple (Malus × domestica 'Royal Gala') that were likely to encode triterpene synthases. Two of these expressed sequence tag sequences were essentially identical (> 99% amino acid similarity; MdOSC1 and MdOSC3). MdOSC1 and MdOSC2 were expressed by transient expression in Nicotiana benthamiana leaves and by expression in the yeast Pichia methanolica. The resulting products were analysed by GC and GC-MS. MdOSC1 was shown to be a mixed amyrin synthase (a 5 : 1 ratio of α-amyrin to β-amyrin). MdOSC1 is the only triterpene synthase so far identified in which the level of α-amyrin produced is > 80% of the total product and is, therefore, primarily an α-amyrin synthase. No product was evident for MdOSC2 when expressed either transiently or in yeast, suggesting that this putative triterpene synthase is either encoded by a pseudogene or does not express well in these systems. Transcript expression analysis in Royal Gala indicated that the genes are mostly expressed in apple peel, and that the MdOSC2 expression level was much lower than that of MdOSC1 and MdOSC3 in all the tissues tested. Amyrin content analysis was undertaken by LC-MS, and demonstrated that levels and ratios differ between tissues, but that the true consequence of synthase activity is reflected in the ursolic/oleanolic acid content and in further triterpenoids derived from them. Phylogenetic analysis placed the three triterpene synthase sequences with other triterpene synthases that encoded either α-amyrin and/or β-amyrin synthase. MdOSC1 and MdOSC3 clustered with the multifunctional triterpene synthases, whereas MdOSC2 was most similar to the β-amyrin synthases.
authors.affiliation: The New Zealand Institute for Plant and Food Research Limited (Plant & Food Research), Auckland, New Zealand.
authors.forename: Cyril
authors.initials: C
authors.lastname: Brendolise
authors.forename: Yar-Khing
authors.initials: YK
authors.lastname: Yauk
authors.forename: Ellen D
authors.initials: ED
authors.lastname: Eberhard
authors.forename: Mindy
authors.initials: M
authors.lastname: Wang
authors.forename: David
authors.initials: D
authors.lastname: Chagne
authors.forename: Christelle
authors.initials: C
authors.lastname: Andre
authors.forename: David R
authors.initials: DR
authors.lastname: Greenwood
authors.forename: Lesley L
authors.initials: LL
authors.lastname: Beuning
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D020224:Expressed Sequence Tags; D005638:Fruit / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant; D019751:Intramolecular Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D027845:Malus / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D008969:Molecular Sequence Data; D009828:Oleanolic Acid / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014315:Triterpenes / Q000032:analysis
chemical_list: D007527:Isoenzymes; D010940:Plant Proteins; D011994:Recombinant Proteins; D014315:Triterpenes; D009828:Oleanolic Acid; D019751:Intramolecular Transferases; C039924:2,3-oxidosqualene-beta-amyrin-cyclase; C021924:lanosterol synthase; C036380:beta-amyrin