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Doctoral Thesis
DOI
https://doi.org/10.11606/T.46.2019.tde-25112019-162943
Document
Author
Full name
Maira Artischeff Frutuoso
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2019
Supervisor
Committee
Marana, Sandro Roberto (President)
Carvalho, Cristiane Rodrigues Guzzo
Salinas, Roberto Kopke
Tersariol, Ivarne Luis dos Santos
Title in Portuguese
Contribuição dos subdomínios (β/α)4 para a estabilidade e atividade de β-glicosidases GH1 com estrutura (β/α)8 barril
Keywords in Portuguese
β-glicosidases GH1
(β/α)8 barril
Subdomínios
Abstract in Portuguese
Domínios são definidos como unidades de dobramento independente e estáveis que formam a estrutura das proteínas (Richardson, 1981; Dootlitle, 1995; Porter e Rose, 2012). Interessantemente quando esta definição é aplicada na análise de proteínas com dobramento (β/α)8 barril identificam-se 2 domínios, que correspondem as suas metades N- e C-terminais (Porter e Rose, 2012), contrastando com a visão que assume o (β/α)8 barril como um domínio único. Deste modo, considerando então que as metades N-terminal e C-terminal das proteínas (β/α)8 barril sejam consideravelmente estáveis e independentes, o objetivo geral desta tese foi avaliar como as propriedades individuais destes subdomínios (β/α)4 se combinam e definem tanto a estabilidade quanto a atividade catalítica das β-glicosidases GH1, que possuem estrutura (β/α)8 barril. As β-glicosidases bglA de Thermotoga marítima (uma bactéria termófila), bglA e bglB de Paenibacillus polymyxa (uma bactéria mesófila) e proteínas quiméricas originadas da combinação dos subdomínios (β/α)4 entre duas destas β-glicosidases (NbglThm-CbglB, NbglB-CbglThm, NbglThm-CbglA, NbglA-CbglThm, NbglA-CbglB e NbglB-CbglA) foram os modelos experimentais. As β-glicosidases bglA, bglB e bglThm foram produzidas como proteínas recombinantes e purificadas. Apenas NbglA-CsbglThm pode ser purificada solúvel e ativa. A fluorescência de triptofanos e o dicroísmo circular sugerem que as β-glicosidases selvagens e a quimera estão enoveladas. Além disso, NbglA-CbglThm apresenta uma composição de estrutura secundária mais próxima à bglA do que bglThm. Por outro lado, o acesso do supressor acrilamida aos triptofanos de NbglA-CbglThm é igual a bglThm e menor do que bglA. A Tm e o Kobs de inativação a 47°C de NbglA-CbglThm são intermediárias àquelas de bglA e bglThm. Portanto, a termoestabilidade da quimera é uma ponderação daquela das suas proteínas parentais. NbglA-CbglThm possui atividade enzimática independente do pH, enquanto bglA e bglThm exibem uma curva em sino. Ainda, NbglA-CbglThm mostrou menor atividade enzimática, ilustrada pela queda dos kcat para os substratos estudados. Considerando que funcionamento do sítio ativo das β-glicosidases depende do posicionamento relativo de pelo menos dez resíduos, estas mudanças na quimera não são inesperadas. NbglA-CbglThm possui Km intermediário para os substratos pNPβ- Gluco e pNPβ-Fuco em relação a bglA e bglThm. Adicionalmente, a especificidade pelo substrato de NbglA-CbglThm é uma ponderação daquela de bglA e bglThm, pois enquanto estas parentais exibiram preferência por um destes substratos, a quimera apresenta o mesmo kcat/Km para os dois. Deste modo, a caracterização da atividade enzimática sugere que as metades (β/α)4 que compõem a quimera NbglA-CbglThm provavelmente se dobraram autossuficientemente e se ajustaram formando um sítio ativo funcional. Coerentemente, a estabilidade térmica intermediária da quimera também sugere que as metades (β/α)4 das proteínas parentais mantiveram sua termoestabilidade relativamente independente. Em conjunto estas observações implicam em razoável estabilidade e independência termodinâmica no dobramento de cada uma das metades (β/α)4, funcionando, portanto, como unidades praticamente independentes, ou seja, como domínios.
Title in English
Contribution of (β/α)4 subdomains to the stability and activity of β-glycosidases GH1 with (β/α)8 barrel structure
Keywords in English
β-glycosidases GH1
(β/α)8 barrel
Subdomains
Abstract in English
Domains are stable and independent folding units that compose the protein structure (Richardson, 1981; Dootlitle, 1995; Porter e Rose, 2012). Interestingly, the application of this definition in the analysis of (β/α)8 barrel proteins reveals two domains corresponding to the N- and C-terminal halves (Porter and Rose, 2012), a perspective that contrast with the common assumption that those are single domain proteins. Based on that, assuming that the N- and C-terminal halves of the (β/α)8 barrel proteins are stable and independent, the objective of this project was to understand how the individual properties of the (β/α)4 subdomains combine and define the stability and catalytic activity of the β-glycosidases GH1, which exhibit (β/α)8 structure. The β-glycosidase bglThm from Thermotoga maritima (thermophile bacteria), bglA and bglB from Paenibacillus polymyxa (mesophile bacteria) and six chimeric proteins combining the (β/ α)4 subdomains from two of those enzymes (NbglThm-CbglB, NbglB-CbglThm, NbglThm-CbglA, NbglA-CbglThm, NbglA-CbglB e NbglB-CbglA) were used as experimental models. bglA, bglB and bglThm were produced as recombinant proteins and successfully purified. The chimera NbglA-CbglThm was the only one that was viably produced and purified. Tryptophan fluorescence and circular dichroism spectra indicated that the wild-type and the chimeric β-glycosidases were folded. NbglA-CbglThm has a secondary structure composition similar to bglA. On the other hand, access of the tryptophan residues of NbglA-CbglThm to the quencher acrylamide is comparable to bglThm. The melting temperature (Tm) and the thermal inactivation rate constant (at 47°C) of the NbglA-CbglThm are intermediary to bglA and bglThm. Hence, the chimera thermostability is a balance of the parental enzymes stabilities. The catalytic activity of NbglA-CbglThm does not depend on the pH, whereas bglA and bglThm exhibited a typical bell shaped curve. In addition, NbglA-CbglThm activity is lower than the parental enzymes, as showed the kcat for three different substrates. Considering that the active site properties of the β-glycosidases rely on the relative positioning of at least ten residues, such changes are not surprising. The Km of NbglA-CbglThm for substrates pNPβ-Gluco and pNPβ-Fuco are intermediary to bglA and bglThm. Finally, the substrate specificity of the NbglA-CbglThm is a balance of the parental enzymes specificities, because they showed clear preference for one of those substrates, whereas the chimera presented the same kcat/Km for both. Hence, the characterization of the chimera activity indicates that the (β/α)4 subdomains folded self-sufficiently and adjusted itself to form a functional active site. In agree, the chimera thermostability also indicates that the (β/α)4 subdomains kept their individual thermal-properties. These observations point to a sufficient thermodynamic stability in the folding and properties of each (β/α)4 half, implying that they were virtually self-contained like true domains.
 
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Publishing Date
2019-12-06
 
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