Avanços no processo de purificação da friedelina sintase e estudos de sua atividade e especificidade
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Data
2019-10-18
Autores
Mazzeu, Bruna Fonseca
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
A engenharia metabólica tem ajudado a melhorar ou redirecionar as vias metabólicas, fazendo modificações genéticas, como, a deleção ou superexpressão de genes e desta forma alterando a atividade de reações enzimáticas específicas. Friedelina é um triterpeno pentacíclico encontrado nas folhas das espécies de Celastraceae, que demonstrou inúmeras atividades biológicas e é precursor dos triterpenos quinonemetideos, promissor antitumoral. É biossintetizada a partir da ciclização do 2,3-oxidosqualeno e envolve o número máximo de rearranjos para a formação de uma cetona por desprotonação do intermediário hidroxilado sem o auxílio de uma enzima oxidoredutase. O estudo das enzimas responsáveis pela produção de metabólitos secundários é importante na busca de novas alternativas sustentáveis para a produção dos mesmos. Nessas condições, friedelina sintase de Maytenus ilicifolia foi clonada e expressa em Saccharomyces cerevisiae e Escherichia coli, levando a sua obtenção em um sistema heterólogo. A obtenção da fração enriquecida com friedelina sintase, foi realizada em duas etapas cromatográficas: cromatografia por afinidade, utilizando uma coluna com resina Ni–NTA (ácido nitrilotriacético) e cromatografia de troca iônica com resina de troca aniônica. A presença e pureza de MiFRS foi avaliada por meio de eletroforese em gel desnaturante de poliacrilamida (SDS-PAGE) revelado com Coomassie e comparado com o perfil em Western Blot. As etapas de purificação removeram uma grande parte das proteínas contaminantes. Os estudos de mutação sitio dirigida in vivo foram realizados com quatro mutações: a troca de um resíduo de cisteína, na posição 369 por um resíduo de prolina (C369P), a troca de uma fenilalanina, na posição 473 por um triptofano (F473W), a troca de um resíduo de metionina, na posição 549 por um resíduo de serina (M549S) e a troca de uma leucina por uma fenilalanina, na posição 552 (L552F). As mutações C369P e F473W levaram a perda de função, além de não produzir qualquer outro triterpeno. Os mutantes M549S e L552F conservaram a atividade da enzima, produzindo friedelina e foram capazes de produzir β-amirina e α-amirina.
Metabolic engineering has helped to improve or redirect metabolic pathways, making genetic modifications such as deletion or overexpression of genes and thus altering the activity of specific enzyme reactions. Friedelin is a pentacyclic triterpene found in the leaves of Celastraceae species, which has demonstrated numerous biological activities and is a precursor of quinonemethide, promising antitumoral. It is biosynthesized from 2,3-oxidosqualene cyclization and involves the maximum number of rearrangements for ketone formation by deprotonation of the hydroxylated intermediate without the aid of an oxidoreductase enzyme. The study of the enzymes responsible for the production of secondary metabolites is important in the search for new sustainable alternatives for their production. Under these conditions, friedelin synthase from Maytenus ilicifolia was cloned and expressed in Saccharomyces cerevisiae and Escherichia coli, leading to its obtention in a heterologous system. Enriched fraction with friedelin synthase was obtained in two chromatographic steps: affinity chromatography using a column with Ni–NTA resin (nitrilotriacetic acid) and ion exchange chromatography with anion exchange resin. The presence and purity of MiFRS was evaluated by polyacrylamide denaturing gel electrophoresis (SDS-PAGE) developed with Coomassie and compared to the Western Blot profile. The purification steps removed a large portion of the contaminating proteins. Site-directed mutation studies were performed with four mutations: exchanging a cysteine residue at position 369 for a proline residue (C369P), exchanging a phenylalanine residue at position 473 for a tryptophan residue (F473W), exchanging a methionine residue at position 549 for a serine residue (M549S) and exchanging a leucine residue for a phenylalanine residue at position 552 (L552F). Mutations C369P and F473W led to loss of function and produced no other triterpene. Mutants M549S and L552F retained enzyme activity, producing friedelin and were capable of producing β-amyrin and α-amyrin.
Metabolic engineering has helped to improve or redirect metabolic pathways, making genetic modifications such as deletion or overexpression of genes and thus altering the activity of specific enzyme reactions. Friedelin is a pentacyclic triterpene found in the leaves of Celastraceae species, which has demonstrated numerous biological activities and is a precursor of quinonemethide, promising antitumoral. It is biosynthesized from 2,3-oxidosqualene cyclization and involves the maximum number of rearrangements for ketone formation by deprotonation of the hydroxylated intermediate without the aid of an oxidoreductase enzyme. The study of the enzymes responsible for the production of secondary metabolites is important in the search for new sustainable alternatives for their production. Under these conditions, friedelin synthase from Maytenus ilicifolia was cloned and expressed in Saccharomyces cerevisiae and Escherichia coli, leading to its obtention in a heterologous system. Enriched fraction with friedelin synthase was obtained in two chromatographic steps: affinity chromatography using a column with Ni–NTA resin (nitrilotriacetic acid) and ion exchange chromatography with anion exchange resin. The presence and purity of MiFRS was evaluated by polyacrylamide denaturing gel electrophoresis (SDS-PAGE) developed with Coomassie and compared to the Western Blot profile. The purification steps removed a large portion of the contaminating proteins. Site-directed mutation studies were performed with four mutations: exchanging a cysteine residue at position 369 for a proline residue (C369P), exchanging a phenylalanine residue at position 473 for a tryptophan residue (F473W), exchanging a methionine residue at position 549 for a serine residue (M549S) and exchanging a leucine residue for a phenylalanine residue at position 552 (L552F). Mutations C369P and F473W led to loss of function and produced no other triterpene. Mutants M549S and L552F retained enzyme activity, producing friedelin and were capable of producing β-amyrin and α-amyrin.
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Palavras-chave
Friedelina sintase, Sistema heterólogo, Modelagem molecular, Mutação sitio dirigida