Determinação das estruturas cristalográficas de epóxido hidrolases de interesse biotecnológico e envolvidas na biossíntese de antibióticos ionóforos
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Data
2017-04-28
Autores
Wilson, Carolina [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
As epóxido hidrolases (EHs) são enzimas que catalisam a conversão de epóxidos em dióis vicinais. Esses compostos são menos reativos, menos mutagênicos, menos tóxicos e são excretados mais rapidamente das células pois apresentam maior solubilidade. Nas reações de catálise, as EHs utilizam a água como o único co-substrato, o que é uma característica particular desta classe enzimática. Em microrganismos, elas atuam na adaptação ao meio ambiente, metabolizando fontes naturais de carbono e contaminantes. Em patógenos, como M. tuberculosis, atuam na virulência e desintoxicação. Além disso, têm importantes aplicações industriais, farmacêuticas e agroquímicas, como a degradação de xenobióticos, separação enantiomérica de misturas de epóxidos, hidrólise regio e enantiosseletiva de de epóxidos quirais e biossíntese de antibióticos poliéteres ionóforos. Os objetivos do presente trabalho são a cristalização e resolução estrutural das epóxido hidrolases B1EPH2 de Streptomyces wadayamensis e TrEH de Trichoderma reesei, que apresentam interesse biotecnológico devido a propriedades enantioseletivas e do mutante Lsd19 E197D (S. lasaliensis) e TsnB (S. longisporoflavius), envolvidas na biossíntese dos antibióticos poliéteres ionóforos lasalocida e tetronasina, respectivamente. Estas enzimas foram co-cristalizadas com diferentes epóxidos, inibidores, substratos e análogos de substrato e as estruturas foram resolvidas por substituição molecular. A B1EPH2 e TrEH apresentaram enovelamento similar às α/β-Hidrolases, com dois domínios distintos, o domínio catalítico conservado ou N-terminal e o domínio cap ou C-terminal. As estruturas de Lsd19 E197D e TsnB apresentaram enovelamento característico das Limoneno Epóxido Hidrolases, com dois domínios – N-terminal e C-terminal – semelhantes entre si e formados por uma β-folha e α-hélices dispostas de forma semelhante a um cone. Além disso, as enzimas do presente estudo foram comparadas a outras EHs com estruturas ja resolvidas no que se refere à presença de resíduos conservados nos sítos ativos. Dessa forma, levantamos algumas hipóteses a respeito de mecanismo catalítico das EHs estudadas.
Epoxide hydrolases (EHs) are enzymes that catalyze the conversion of epoxides to vicinal diols. These compounds are less reactive, less mutagenic, less toxic and are excreted more rapidly from the cells because they have greater solubility. In the catalysis reactions, the EHs use water as the only co-substrate, which is a particular characteristic of this enzymatic class. In microorganisms, they act in the adaptation to the environment, metabolizing natural sources of carbon and contaminants. In pathogens, such as M. tuberculosis, they act on virulence and detoxification. In addition, they have important industrial, pharmaceutical and agrochemical applications, such as degradation of xenobiotics, enantiomeric separation of epoxide mixtures, regio and enantioselective hydrolysis of chiral epoxides and biosynthesis of ionophores polyethers antibiotics. The objectives of the present work are the crystallization and structural resolution of the epoxide hydrolases B1EPH2 from Streptomyces wadayamensis and TrEH from Trichoderma reesei, which show biotechnological interest due to enantioselective properties and the mutant Lsd19 E197D (S. lasaliensis) and TsnB (S. longisporoflavius), involved in the biosynthesis of the ionophores antibiotics lasalocid and tetronasin, respectively. These enzymes were co-crystallized with different epoxides, inhibitors, substrates and substrate analogues and the structures were resolved by molecular substitution. B1EPH2 and TrEH showed similar folding to α/β-hydrolases, with two distinct domains, the conserved or N-terminal catalytic domain and the cap or C-terminal domain. The structures of Lsd19 E197D and TsnB presented a characteristic folding of Limonene Epoxide Hydrolases, with two domains – N-terminal and C-terminal – similar to each other and formed by a β-sheet and α-helices arranged similarly to a cone. In addition, the enzymes of the present study were compared to other EHs with structures already resolved with respect to the presence of residues conserved in the active sites. Thus, we raise some hypotheses about the catalytic mechanism of the EHs studied.
Epoxide hydrolases (EHs) are enzymes that catalyze the conversion of epoxides to vicinal diols. These compounds are less reactive, less mutagenic, less toxic and are excreted more rapidly from the cells because they have greater solubility. In the catalysis reactions, the EHs use water as the only co-substrate, which is a particular characteristic of this enzymatic class. In microorganisms, they act in the adaptation to the environment, metabolizing natural sources of carbon and contaminants. In pathogens, such as M. tuberculosis, they act on virulence and detoxification. In addition, they have important industrial, pharmaceutical and agrochemical applications, such as degradation of xenobiotics, enantiomeric separation of epoxide mixtures, regio and enantioselective hydrolysis of chiral epoxides and biosynthesis of ionophores polyethers antibiotics. The objectives of the present work are the crystallization and structural resolution of the epoxide hydrolases B1EPH2 from Streptomyces wadayamensis and TrEH from Trichoderma reesei, which show biotechnological interest due to enantioselective properties and the mutant Lsd19 E197D (S. lasaliensis) and TsnB (S. longisporoflavius), involved in the biosynthesis of the ionophores antibiotics lasalocid and tetronasin, respectively. These enzymes were co-crystallized with different epoxides, inhibitors, substrates and substrate analogues and the structures were resolved by molecular substitution. B1EPH2 and TrEH showed similar folding to α/β-hydrolases, with two distinct domains, the conserved or N-terminal catalytic domain and the cap or C-terminal domain. The structures of Lsd19 E197D and TsnB presented a characteristic folding of Limonene Epoxide Hydrolases, with two domains – N-terminal and C-terminal – similar to each other and formed by a β-sheet and α-helices arranged similarly to a cone. In addition, the enzymes of the present study were compared to other EHs with structures already resolved with respect to the presence of residues conserved in the active sites. Thus, we raise some hypotheses about the catalytic mechanism of the EHs studied.
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Palavras-chave
Epóxido hidrolases, Enantiosseletividade, Poliéteres ionóforos, Produtos naturais, Cristalografia, Epoxide hydrolases, Enantioselectivity, Ionophores polyethers, Natural products, Crystallography