Estudos estruturais e funcionais de duas glicosideo hidrolases : a celulase putativa XF0810 de Xylella fastidiosa e a lisozima digestiva 1 de Musca domestica
Amanda Abdalla Valerio
TESE
Português
(Broch.)
T/UNICAMP V237e
[Structural and functional studies of two glycosyl hydrolases]
Campinas, SP : [s.n.], 2007.
141p. : il.
Orientador: João Alexandre Ribeiro Gonçalves Barbosa
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
Resumo: Glicosídeo hidrolases (EC 3.2.1.-) são enzimas que hidrolisam ligações glicosídicas. Neste trabalho realizamos estudos funcionais e estruturais de duas glicosídeo hidrolases: a celulase XF0810 de Xylella fastidiosa e a lisozima digestiva 1 de Musca domestica (MdL1). A celulase XF0810 está...
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Resumo: Glicosídeo hidrolases (EC 3.2.1.-) são enzimas que hidrolisam ligações glicosídicas. Neste trabalho realizamos estudos funcionais e estruturais de duas glicosídeo hidrolases: a celulase XF0810 de Xylella fastidiosa e a lisozima digestiva 1 de Musca domestica (MdL1). A celulase XF0810 está anotada no genoma da X. fastidiosa como membro da família 5 das glicosídeo hidrolases (EC 3.2.1.4). Após a amplificação, clonagem, expressão e purificação da mesma; prosseguimos com os experimentos de filtração em gel analítica, dicroísmo circular, DLS, ensaio enzimático e cristalização desta proteína. Foi feito um estudo de modelagem onde ficou evidenciado que a XF0810 não pertenceria à família 5 pois quatro dos sete resíduos conservados que caracterizam esta família foram substituídos, incluindo os dois resíduos catalíticos de glutamato essenciais para o mecanismo de clivagem de retenção. Isto foi comprovado através da ausência de atividade nos ensaios feitos com a proteína purificada. A MdL1 pertence à família 22 das glicosídeo hidrolases (EC 3.2.1.17) e foi cristalizada com o ligante Nacetilquitotetraosídeo para a difração de raios X. A resolução da estrutura (2H5Z no PDB) foi realizada por meio do método de substituição molecular tendo como modelo a estrutura nativa. A análise comparativa da MdL1 com outras lisozimas de quatro classes diferentes de animais mostrou grande semelhança e pequenas diferenças apenas na região das voltas. Estas diferenças foram utilizadas para explicar as características especiais de uma lisozima com função digestiva. A volta na região definida pelos resíduos 98-100 apresenta uma deleção na MdL1, tornado-a menos exposta ao solvente, podendo justificar a resistência à proteólise. O resíduo Gln100 participa de uma interação com o ligante. Os resíduos Thr107 e Asn46 são apontados como responsáveis pelo decréscimo dos pKa dos grupos carboxilas dos resíduos catalíticos Glu32 e Asp50, respectivamente. A diminuição dos pKa explica o pH ótimo mais ácido característico de lisozimas digestivas
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Abstract: Glycoside hydrolases (EC 3.2.1.-) are enzymes that hydrolyze glycoside bonds. In this work we studied functional and structural features of two glycoside hydrolases: the cellulase XF0810 of Xylella fastidiosa and the digestive lysozyme 1 of Musca domestica (MdL1). The cellulase XF0810 is...
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Abstract: Glycoside hydrolases (EC 3.2.1.-) are enzymes that hydrolyze glycoside bonds. In this work we studied functional and structural features of two glycoside hydrolases: the cellulase XF0810 of Xylella fastidiosa and the digestive lysozyme 1 of Musca domestica (MdL1). The cellulase XF0810 is annotated in the genome of
X. fastidiosa as member of family 5 of glycoside hydrolases (EC 3.2.1.4). After the amplification, cloning, expression and purification of XF0810; we continued with the experiments of analytical gel filtration, circular dichroism, DLS, enzymatic assay and crystallization of this protein. A homology model was built which showed that XF0810 did not belong to family 5 because four of seven conserved residues that characterize the family were substituted, including the two catalytic residues of glutamate that are essential for the retention hydrolysis mechanism. This was further confirmed by the absence of activity in the assays (exocellulase with PNPc) performed with the purified protein. The MdL1 belongs to the family 22 of glycoside hydrolases (EC 3.2.1.17) and was crystallized with the ligand N-acetilchitotetraose for X-ray diffraction. The resolution of the structure (2H5Z in PDB) was accomplished by molecular replacement with the native structure as the searching model. The comparative analysis of MdL1 with other lysozymes of four different classes of animals showed a high similarity and few differences appeared only in the loops¿ regions. These differences were used to explain the special characteristics of the lysozyme with a digestive function. The loop in the region defined by residues 98-100 presents one deletion in the MdL1, becoming less exposed to the solvent, this might justify the proteolysis resistance. The residue Gln100 participates directly in an interaction with the ligand. The residues Thr107 and Asn46 are pointed out as responsible for a reduction in the pKa of the carboxyl groups of catalytic residues Glu32 e Asp50, respectively. The reduction in pKa explains the more acidic pH optimum that characterizes the digestive lysozymes Ver menos
X. fastidiosa as member of family 5 of glycoside hydrolases (EC 3.2.1.4). After the amplification, cloning, expression and purification of XF0810; we continued with the experiments of analytical gel filtration, circular dichroism, DLS, enzymatic assay and crystallization of this protein. A homology model was built which showed that XF0810 did not belong to family 5 because four of seven conserved residues that characterize the family were substituted, including the two catalytic residues of glutamate that are essential for the retention hydrolysis mechanism. This was further confirmed by the absence of activity in the assays (exocellulase with PNPc) performed with the purified protein. The MdL1 belongs to the family 22 of glycoside hydrolases (EC 3.2.1.17) and was crystallized with the ligand N-acetilchitotetraose for X-ray diffraction. The resolution of the structure (2H5Z in PDB) was accomplished by molecular replacement with the native structure as the searching model. The comparative analysis of MdL1 with other lysozymes of four different classes of animals showed a high similarity and few differences appeared only in the loops¿ regions. These differences were used to explain the special characteristics of the lysozyme with a digestive function. The loop in the region defined by residues 98-100 presents one deletion in the MdL1, becoming less exposed to the solvent, this might justify the proteolysis resistance. The residue Gln100 participates directly in an interaction with the ligand. The residues Thr107 and Asn46 are pointed out as responsible for a reduction in the pKa of the carboxyl groups of catalytic residues Glu32 e Asp50, respectively. The reduction in pKa explains the more acidic pH optimum that characterizes the digestive lysozymes Ver menos
Barbosa, João Alexandre Ribeiro Gonçalves
Orientador
Benedetti, Celso Eduardo, 1964-
Avaliador
Kobarg, Jörg, 1965-
Avaliador
Garratt, Richard Charles
Avaliador
Freitas, Sonia Maria de
Avaliador
Estudos estruturais e funcionais de duas glicosideo hidrolases : a celulase putativa XF0810 de Xylella fastidiosa e a lisozima digestiva 1 de Musca domestica
Amanda Abdalla Valerio
Estudos estruturais e funcionais de duas glicosideo hidrolases : a celulase putativa XF0810 de Xylella fastidiosa e a lisozima digestiva 1 de Musca domestica
Amanda Abdalla Valerio
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