Oxidoredutase de Maytenus: seu papel na biossíntese dos derivados friedelânicos
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Data
2022-07-06
Autores
Anhesine, Naira Buzzo [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A espécie Maytenus ilicifolia, Celastraceae, é popularmente conhecida como “espinheira-santa” e suas folhas são amplamente utilizadas na forma de chá na medicina popular para o tratamento de úlceras e gastrites estomacais. Estudos químicos e biológicos relatam outras atividades tais como contraceptiva, analgésica, anti-inflamatória, antipirética, antioxidante entre outras, que estão intimamente relacionadas com a presença de flavonoides, taninos e terpenos. Os triterpenos pentacíclicos (C30) é a subclasse que merece destaque em espécies do gênero Maytenus, dentre eles a friedelina e friedelan-3-ol, os quais são as substâncias mais abundantes no extrato de baixa polaridade das folhas de M. ilicifolia. A friedelina é uma substância de grande interesse terapêutico, pois além de ser precursora de substâncias com potencial antitumoral tais como maitenina e pristimerina, ambos terpenos quinonametídeos, ela apresenta atividades biológicas intrínsecas atuando como anti-inflamatório, analgésico, antipirético, antimicrobiano e gastroprotetor. Do ponto de vista biossintético, a friedelina é o único triterpeno pentacíclico que se apresenta oxidado no carbono 3 (C-3) como cetona e o mecanismo proposto de formação desta função não passa pela formação de uma forma enólica. No entanto, espécies de Celastraceae, especialmente do gênero Maytenus, mostram, nas suas partes aéreas, o acúmulo concomitante de friedelina e seu derivado reduzido, friedelan-3-ol, suscitando assim, interessantes aspectos biossintéticos a serem investigados. Neste contexto, o objetivo é identificar e caracterizar uma enzima do tipo redutase, que a princípio ainda não foi identificada em plantas, capaz de reduzir um grupo cetônico na posição C-3 de terpenos em seus derivados alcoólicos utilizando ferramentas da biologia molecular e microbiologia. A abordagem empregada consistiu em construir uma base de dados de enzimas 3-ceto redutase, uma subclasse de “Short-Chain Dehydrogenase/Reductase (SDR)”, cuja função enzimática é oxidoredutase atuando em C-3 em rotas biossintéticas de fungos (ERG27) e vertebrados (HSD17B7), e alinhá-la aos transcriptomas de M. ilicifolia e de Quercus suber, espécie igualmente rica em friedelina e friedelan-3-ol. Posteriormente, foi realizada análise cruzada de ambos os transcriptomas para filtrar os transcritos em comum resultando em três promissores: 45855_i6, 52338_i1 e 52338_i6. Eles foram clonados por tecnologia Gateway e transformados em levedura Saccharomyces cerevisiae geneticamente modificada juntamente com ogene da friedelina sintase cuja função era fornecer o substrato (friedelina) para a enzima oxidoredutase de interesse. A expressão heteróloga destes transcritos e a análise dos metabólitos extraídos por CG-EM sugerem que os mesmos não sejam os responsáveis pela redução em C-3 uma vez que não foi observado a presença de friedelan-3-ol nos cromatogramas. De qualquer forma, não se exclui a possibilidade de que outro transcrito presente no transcriptoma possa ser o responsável por essa redução, seja ele codificador de uma enzima do tipo 3-ceto redutase ou de outra subclasse de SDR. Acredita-se que exista sim uma enzima em plantas que seja homóloga ao ERG27 em fungos e ao HSD17B7 em vertebrados que faz essa redução em C-3 e o presente estudo já é um grande material de partida para pesquisas futuras que têm como objetivo a busca por tal enzima.
Maytenus ilicifolia, Celastraceae, is popularly known as “espinheira-santa” and its leaves are used as tea in folk medicine for the treatment of stomach ulcers and gastritis. Chemical and biological studies described other activities such as contraceptive, analgesic, anti-inflammatory, antipyretic, antioxidant, among others, which are closely related to the presence of flavonoids, tannins and terpenes. Pentacyclic triterpenes (C30) is a subclass that deserves to be highlighted in species of the genus Maytenus, including friedelin and friedelan-3-ol, which are the most abundant compounds in the non-polar extract of M. ilicifolia leaves. Friedelin is a compound of great therapeutic interest, as in addition to being a precursor of substances with antitumor potential such as maytenin and pristimerin, both quinonemethide triterpenes, it has intrinsic biological activities acting as antiinflammatory, analgesic, antipyretic, antimicrobial and gastroprotective. From a biosynthetic point of view, friedelin is the only pentacyclic triterpene that is oxidized at carbon 3 (C-3) as a ketone and the proposed mechanism for the formation of this function does not involve the formation of an enol form. However, Celastraceae species, especially of the Maytenus genus, show, in their aerial parts, the concomitant accumulation of friedelin and its reduced derivative, friedelan-3-ol, thus raising interesting biosynthetic aspects to be investigated. In this context, this project aims to identify and characterize a reductase-like enzyme, which hasn’t been identified in plantas yet, that is capable of reducing a ketone group at the C-3 position of terpenes in its alcoholic derivatives using molecular biology and microbiology tools. The approach considered in the present work consisted of building a database of 3-keto reductase enzymes, a subclass of “Short-Chain Dehydrogenase/Reductase (SDR)” whose enzymatic function is C-3 oxidoreductase in fungal (ERG27) and vertebrates (HS17B7) biosynthetic pathways, and aligning it to the transcriptomes of M. ilicifolia and Quercus suber, a species equally rich in friedelin and friedelan-3-ol. Subsequently, cross analysis of both transcriptomes was performed to filter the transcripts in common, resulting in three promising ones: 45855_i6, 52338_i1 and 52338_i6. They were cloned by Gateway technology and transformed into genetically modified yeast Saccharomyces cerevisiae together with the friedelin synthase gene whose function was to provide the substrate (friedelin) for the enzyme of interest. A heterologous expression of these transcripts and an analysis of the extractedmetabolites by GC-MS suggested that they are not responsible for the reduction in C- 3 since the presence of friedelan-3-ol in the chromatograms was not observed. In any case, it is not excluded the possibility that another transcript may be responsible for this reduction, whether encoding an enzyme of the 3-ketoreductase type or another subclass of SDR. It is believed that there is indeed an enzyme in plants that is homologous to ERG27 in fungi and to HSD17B7 in vertebrates that makes this reduction in C-3 and the present study is already a great starting material for future research aimed at the search by such an enzyme.
Maytenus ilicifolia, Celastraceae, is popularly known as “espinheira-santa” and its leaves are used as tea in folk medicine for the treatment of stomach ulcers and gastritis. Chemical and biological studies described other activities such as contraceptive, analgesic, anti-inflammatory, antipyretic, antioxidant, among others, which are closely related to the presence of flavonoids, tannins and terpenes. Pentacyclic triterpenes (C30) is a subclass that deserves to be highlighted in species of the genus Maytenus, including friedelin and friedelan-3-ol, which are the most abundant compounds in the non-polar extract of M. ilicifolia leaves. Friedelin is a compound of great therapeutic interest, as in addition to being a precursor of substances with antitumor potential such as maytenin and pristimerin, both quinonemethide triterpenes, it has intrinsic biological activities acting as antiinflammatory, analgesic, antipyretic, antimicrobial and gastroprotective. From a biosynthetic point of view, friedelin is the only pentacyclic triterpene that is oxidized at carbon 3 (C-3) as a ketone and the proposed mechanism for the formation of this function does not involve the formation of an enol form. However, Celastraceae species, especially of the Maytenus genus, show, in their aerial parts, the concomitant accumulation of friedelin and its reduced derivative, friedelan-3-ol, thus raising interesting biosynthetic aspects to be investigated. In this context, this project aims to identify and characterize a reductase-like enzyme, which hasn’t been identified in plantas yet, that is capable of reducing a ketone group at the C-3 position of terpenes in its alcoholic derivatives using molecular biology and microbiology tools. The approach considered in the present work consisted of building a database of 3-keto reductase enzymes, a subclass of “Short-Chain Dehydrogenase/Reductase (SDR)” whose enzymatic function is C-3 oxidoreductase in fungal (ERG27) and vertebrates (HS17B7) biosynthetic pathways, and aligning it to the transcriptomes of M. ilicifolia and Quercus suber, a species equally rich in friedelin and friedelan-3-ol. Subsequently, cross analysis of both transcriptomes was performed to filter the transcripts in common, resulting in three promising ones: 45855_i6, 52338_i1 and 52338_i6. They were cloned by Gateway technology and transformed into genetically modified yeast Saccharomyces cerevisiae together with the friedelin synthase gene whose function was to provide the substrate (friedelin) for the enzyme of interest. A heterologous expression of these transcripts and an analysis of the extractedmetabolites by GC-MS suggested that they are not responsible for the reduction in C- 3 since the presence of friedelan-3-ol in the chromatograms was not observed. In any case, it is not excluded the possibility that another transcript may be responsible for this reduction, whether encoding an enzyme of the 3-ketoreductase type or another subclass of SDR. It is believed that there is indeed an enzyme in plants that is homologous to ERG27 in fungi and to HSD17B7 in vertebrates that makes this reduction in C-3 and the present study is already a great starting material for future research aimed at the search by such an enzyme.
Descrição
Palavras-chave
Maytenus, Espinheira Santa, Biossíntese, Terpenos, Transcriptoma