title: Friedelin Synthase from Maytenus ilicifolia: Leucine 482 Plays an Essential Role in the Production of the Most Rearranged Pentacyclic Triterpene.
abstract: Among the biologically active triterpenes, friedelin has the most-rearranged structure produced by the oxidosqualene cyclases and is the only one containing a cetonic group. In this study, we cloned and functionally characterized friedelin synthase and one cycloartenol synthase from Maytenus ilicifolia (Celastraceae). The complete coding sequences of these 2 genes were cloned from leaf mRNA, and their functions were characterized by heterologous expression in yeast. The cycloartenol synthase sequence is very similar to other known OSCs of this type (approximately 80% identity), although the M. ilicifolia friedelin synthase amino acid sequence is more related to β-amyrin synthases (65-74% identity), which is similar to the friedelin synthase cloned from Kalanchoe daigremontiana. Multiple sequence alignments demonstrated the presence of a leucine residue two positions upstream of the friedelin synthase Asp-Cys-Thr-Ala-Glu (DCTAE) active site motif, while the vast majority of OSCs identified so far have a valine or isoleucine residue at the same position. The substitution of the leucine residue with valine, threonine or isoleucine in M. ilicifolia friedelin synthase interfered with substrate recognition and lead to the production of different pentacyclic triterpenes. Hence, our data indicate a key role for the leucine residue in the structure and function of this oxidosqualene cyclase.
authors.affiliation: Instituto de Química, Univ. Estadual Paulista-UNESP, Rua Prof. Francisco Degni, 55, Quitandinha, Araraquara, SP 14800-060, Brazil.
authors.forename: Tatiana M
authors.initials: TM
authors.lastname: Souza-Moreira
authors.affiliation: Instituto de Química, Univ. Estadual Paulista-UNESP, Rua Prof. Francisco Degni, 55, Quitandinha, Araraquara, SP 14800-060, Brazil.
authors.forename: Thaís B
authors.initials: TB
authors.lastname: Alves
authors.affiliation: Instituto de Química, Univ. Estadual Paulista-UNESP, Rua Prof. Francisco Degni, 55, Quitandinha, Araraquara, SP 14800-060, Brazil.
authors.forename: Karina A
authors.initials: KA
authors.lastname: Pinheiro
authors.affiliation: Instituto de Química, Univ. Estadual Paulista-UNESP, Rua Prof. Francisco Degni, 55, Quitandinha, Araraquara, SP 14800-060, Brazil.
authors.forename: Lidiane G
authors.initials: LG
authors.lastname: Felippe
authors.affiliation: Centro de Pesquisa e Inovação em Biodiversidade e Fármacos, Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, SP 13563-120, Brazil.
authors.forename: Gustavo M A
authors.initials: GM
authors.lastname: De Lima
authors.affiliation: Instituto de Química, Univ. Estadual Paulista-UNESP, Rua Prof. Francisco Degni, 55, Quitandinha, Araraquara, SP 14800-060, Brazil.
authors.forename: Tatiana F
authors.initials: TF
authors.lastname: Watanabe
authors.affiliation: Faculdade de Ciências Farmacêuticas, Univ. Estadual Paulista-UNESP, Rod. Araraquara-Jaú km 1, Araraquara, SP 14801-902, Brazil.
authors.forename: Cristina C
authors.initials: CC
authors.lastname: Barbosa
authors.affiliation: Instituto de Química, Univ. Estadual Paulista-UNESP, Rua Prof. Francisco Degni, 55, Quitandinha, Araraquara, SP 14800-060, Brazil.
authors.forename: Vânia A F F M
authors.initials: VA
authors.lastname: Santos
authors.affiliation: Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, Avenida do Café s/n, Monte Alegre, Ribeirão Preto, SP 14040-903, Brazil.
authors.forename: Norberto P
authors.initials: NP
authors.lastname: Lopes
authors.affiliation: Faculdade de Ciências Farmacêuticas, Univ. Estadual Paulista-UNESP, Rod. Araraquara-Jaú km 1, Araraquara, SP 14801-902, Brazil.
authors.forename: Sandro R
authors.initials: SR
authors.lastname: Valentini
authors.affiliation: Centro de Pesquisa e Inovação em Biodiversidade e Fármacos, Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, SP 13563-120, Brazil.
authors.forename: Rafael V C
authors.initials: RV
authors.lastname: Guido
authors.affiliation: Instituto de Química, Univ. Estadual Paulista-UNESP, Rua Prof. Francisco Degni, 55, Quitandinha, Araraquara, SP 14800-060, Brazil.
authors.forename: Maysa
authors.initials: M
authors.lastname: Furlan
authors.affiliation: Faculdade de Ciências Farmacêuticas, Univ. Estadual Paulista-UNESP, Rod. Araraquara-Jaú km 1, Araraquara, SP 14801-902, Brazil.
authors.forename: Cleslei F
authors.initials: CF
authors.lastname: Zanelli
mesh_terms: D020816:Amino Acid Motifs; D001665:Binding Sites; D020134:Catalytic Domain; D019751:Intramolecular Transferases / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D007930:Leucine / Q000737:chemistry / Q000378:metabolism; D029755:Maytenus / Q000201:enzymology* / Q000235:genetics; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D009828:Oleanolic Acid / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D010802:Phylogeny; D018515:Plant Leaves / Q000235:genetics; D010940:Plant Proteins / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D018749:RNA, Plant / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D014315:Triterpenes / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D000094042:Lupanes
chemical_list: D019751:Intramolecular Transferases; D007930:Leucine; D009828:Oleanolic Acid; D010940:Plant Proteins; D018749:RNA, Plant; D014315:Triterpenes; C060796:friedelin; D000094042:Lupanes; C039924:2,3-oxidosqualene-beta-amyrin-cyclase; C021924:lanosterol synthase; C021923:cycloartenol synthase; C036380:beta-amyrin