Elucidação estrutural da especificidade por substratos de enzimas da biossíntese de aminoglicosídeos e marginolactonas
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2020-06-26
Autores
Cardoso, Tábata Peres
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Universidade Estadual Paulista (Unesp)
Resumo
Os produtos naturais desempenham um papel importante no desenvolvimento de medicamentos e parte desse sucesso está relacionado com a diversidade química e estrutural apresentadas pelos compostos dotados de relevância biológica. Um exemplo de produtos naturais oriundos do metabolismo secundário de microrganismos são os antibióticos aminoglicosídicos e policetídicos. A complexidade estrutural implica em dificuldades para produzir análogos com atividades medicinais melhoradas. Por outro lado, a biossíntese combinatorial explora a promiscuidade de substratos e emprega enzimas e vias modificadas para produzir novos produtos "não naturais". No presente trabalho, buscamos elucidar estruturalmente enzimas envolvidas na biossíntese de aminoglicosídeos (NeoB, NeoQ, ParB, ParQ, LivB e LivQ) e policetídeos marginolactonas (AMH_821, AMH_828, Medi_4948) com aplicação biotecnológicas. Para isso, genes foram clonados e expressos em Escherichia coli e as proteínas recombinantes foram purificadas. Para as proteínas NeoQ e ParQ, foram realizados teste de expressão a fim de encontrar uma condição de maior rendimento proteico. Para a proteína NeoB, foi realizado um ensaio de deslocamento térmico para escolha do melhor tampão para purificação. Cristais das proteínas NeoB, AMH_828, Medi_4948 foram obtidos em diferentes condições. Aplicamos métodos de resolução estrutural por substituição molecular para todas as proteínas estudadas. Foram resolvidas as estruturas das proteínas NeoB e Medi_4948. A estrutura da enzimaMedi_4948 possui um enovelamento característicos de α/β hidrolases, compartilhado por ureohidrolases e foi possivel observar que ela é hexamêrica, composta por dois trímeros. Esta proteína contém uma alça flexível na região do N-terminal o qual interage com a unidade subjacente do monomero vizinho. O sítio ativo apresenta um cluster binuclear para um íon metal, o qual, juntamente com uma molécula de água é importante para a catálise. A enzima NeoB revelou uma estrutura que contém contém 12 fitas-β e 11 α-hélices. Sete fitas-β formam uma folha-β mista que é bem característico dos membros da superfamília das aspartato aminotransferases. Foi possível também observar um motivo grampo β. Nós mostramos as primeiras informações estruturais obtidas sobre a amidinohidrolase Medi4948 e também conseguimos resultados para outra enzima, uma aminotransferase da biossíntese de neomicina, Neo B.
Natural products play an important role in the development of drugs and part of this success is related to the chemical and structural diversity of compounds with biological relevance. Examples of natural products derived from the secondary metabolism of microorganisms are the aminoglycoside and polyketide antibiotics. Structural complexity, in many cases, makes it difficult to produce analogues with improved pharmacological proprieties. Combinatorial biosynthesis explores the promiscuity of substrates and makes use of enzymes and modified pathways to produce new "unnatural" products, substantially increasing the structural diversity of natural products with potential pharmaceutical value. In this work, we aimed to elucidate structures of enzymes involved in the aminoglycoside and polyketide biosynthesis with biosynthetic applications. NeoB, NeoQ, ParB, ParQ, LivB and LivQ for aminoglycoside and AMH_821, AMH_828, and Medi_4948 for polyketide biosyntheses. Several genes were cloned and expressed in Escherichia coli and the recombinant proteins were purified. For the NeoQ and ParQ proteins, expression tests were performed in order to identify a condition with a higher yield protein production. In addition, for NeoB, thermal shift test was performed to choose the best buffer for purification. Crystals from NeoB proteins, AMH_828, Medi_4948 were obtained under different conditions. We apply structural resolution methods by molecular substitution to all studied proteins. The structures of the NeoB and Medi_4948 proteins were resolved. The structure of the enzymeMedi_4948 has a folding characteristic of α/β hydrolases, shared by ureohydrolases and it was possible to observe that it is hexameric, composed of two trimers. This protein contains a flexible loop in the N-terminal region which interacts with the adjacent monomer. The active site has a binuclear cluster for a metal ion, which, together with a water molecule, is important for catalysis. NeoB enzyme revealed a structure that contains 12 β-tapes and 11 α-helices. Seven β-strands form a mixed β-sheet that is very characteristic in the members of the aspartate aminotransferase superfamily. It was also possible to observe a β clamp motif. We show the first structural information obtained about the amidinohydrolase Medi_4948 and also obtained results for another enzyme, a neomycin biosynthesis aminotransferase, NeoB.
Natural products play an important role in the development of drugs and part of this success is related to the chemical and structural diversity of compounds with biological relevance. Examples of natural products derived from the secondary metabolism of microorganisms are the aminoglycoside and polyketide antibiotics. Structural complexity, in many cases, makes it difficult to produce analogues with improved pharmacological proprieties. Combinatorial biosynthesis explores the promiscuity of substrates and makes use of enzymes and modified pathways to produce new "unnatural" products, substantially increasing the structural diversity of natural products with potential pharmaceutical value. In this work, we aimed to elucidate structures of enzymes involved in the aminoglycoside and polyketide biosynthesis with biosynthetic applications. NeoB, NeoQ, ParB, ParQ, LivB and LivQ for aminoglycoside and AMH_821, AMH_828, and Medi_4948 for polyketide biosyntheses. Several genes were cloned and expressed in Escherichia coli and the recombinant proteins were purified. For the NeoQ and ParQ proteins, expression tests were performed in order to identify a condition with a higher yield protein production. In addition, for NeoB, thermal shift test was performed to choose the best buffer for purification. Crystals from NeoB proteins, AMH_828, Medi_4948 were obtained under different conditions. We apply structural resolution methods by molecular substitution to all studied proteins. The structures of the NeoB and Medi_4948 proteins were resolved. The structure of the enzymeMedi_4948 has a folding characteristic of α/β hydrolases, shared by ureohydrolases and it was possible to observe that it is hexameric, composed of two trimers. This protein contains a flexible loop in the N-terminal region which interacts with the adjacent monomer. The active site has a binuclear cluster for a metal ion, which, together with a water molecule, is important for catalysis. NeoB enzyme revealed a structure that contains 12 β-tapes and 11 α-helices. Seven β-strands form a mixed β-sheet that is very characteristic in the members of the aspartate aminotransferase superfamily. It was also possible to observe a β clamp motif. We show the first structural information obtained about the amidinohydrolase Medi_4948 and also obtained results for another enzyme, a neomycin biosynthesis aminotransferase, NeoB.
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Palavras-chave
Enzimas, Estrutura, Aminoglicosídeos, Marginolactonas, Enzymes, Aminoglycoside antibiotics, Marginolactone, Structure