Enzymes are today well recognized in various industrial applications, being an important component in detergents, and catalysts in the production of agrochemicals, foods, pharmaceuticals, and fine chemicals. Their large use is mainly due to their high selectivity and environmental advantage, compared to traditional catalysts. Tools and techniques in molecular biology offer the possibility to screen the natural sources and engineer new enzyme activities which further increases their usefulness as catalysts, in a broader area. Although enzymes show high substrate and reaction selectivity many enzymes are today known to catalyze other reactions than their natural ones. This is called enzyme promiscuity. It has been suggested that enzyme promiscuity is Nature’s way to create diversity. Small changes in the protein sequence can give the enzyme new reaction specificity. In this thesis I will present how rational design, based on molecular modeling, can be used to explore enzyme promiscuity and to change the enzyme reaction specificity. The first part of this work describes how Candida antarctica lipase B (CALB), by a single point mutation, was mutated to give increased activity for aldol additions, Michael additions and epoxidations. The activities of these reactions were predicted by quantum chemical calculations, which suggested that a single-point mutant of CALB would catalyze these reactions. Hence, the active site of CALB, which consists of a catalytic triad (Ser, His, Asp) and an oxyanion hole, was targeted by site-directed mutagenesis and the nucleophilic serine was mutated for either glycine or alanine. Enzymes were expressed in Pichia pastoris and analyzed for activity of the different reactions. In the case of the aldol additions the best mutant showed a four-fold initial rate over the wild type enzyme, for hexanal. Also Michael additions and epoxidations were successfully catalyzed by this mutant. In the last part of this thesis, rational design of alanine racemase from Geobacillus stearothermophilus was performed in order to alter the enzyme specificity. Active protein was expressed in Escherichia coli and analyzed. The explored reaction was the conversion of alanine to pyruvate and 2-butanone to 2-butylamine. One of the mutants showed increased activity for transamination, compared to the wild type. / QC 20100929

Made available in DSpace on 2017-07-20T12:40:20Z (GMT). No. of bitstreams: 1 Agnes Thiane Machado.pdf: 7176772 bytes, checksum: 01a4049f5c4aed0935803a0cf3a6468d (MD5) Previous issue date: 2017-03-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Study of protein three-dimensional structures allow us to investigate the relations between amino acid sequence, structure and function, what is important chiefly for proteins from pathogenic organisms or ones that belong to the same genus of these, such that they can be used as a structural model. In this context, this work aims at the structural characterization of the enzymes alanine racemase long isoform from Trypanosoma cruzi and glyceraldehyde-3-phosphate dehydrogenase from Naegleria gruberi. The long isoform of alanine racemase catalyzes the conversion between L and D-alanine which, in turn, is part of one of the metabolic pathways in Trypanosoma cruzi, the etiologic agent of Chagas disease. The heterologous expression of this enzyme in Escherichia coli BL21 (DE3) GroEL was analyzed by SDS-PAGE, which revealed that the protein is in higher proportion in the insoluble fraction, thus it was necessary to establish a recovery protocol followed by an in vitro refolding. Data from enzymatic assays and circular dichroism revealed the success of the recovery/refolding protocol, which may in the future contribute to the search for specific inhibitors. Glyceraldehyde-3-phosphate dehydrogenase from Naegleria gruberi catalyzes the sixth step of the organism’s glycolytic pathway. NgGAPDH enzyme was expressed in E. coli (DE3) using the pET-15b vector, and then purified by tree chromatographic steps, two of nickel affinity and one of size exclusion. The enzymatic characterization was investigated with the enzyme without the his-tag; NgGAPDH presented higher activity at pH 8.0, 25 °C and 10 mM of arsenate, and positive cooperativity for substrates G3P and NAD+. His-tag depleted NgGAPDH crystals appeared in 3 days after drop settings, the best crystal diffracted to 1.94 A resolution and belongs to space group P21 with cell parameters a = 83.74 A, b = 94.55 A, c = 90.93 A, = 99.96 °. The final refined structure presents R = 0.1652 and Rfree = 0.2029. The catalytic domain formed by residues 134 to 313 is highly conserved, as expected, with the exception of Asn145, present only in NgGAPDH, while the other GAPDHs present either Ser or Thr on the corresponding position. Molecular dynamics analysis revealed that Asn145 has correlated motion with residues Ala123, Thr125 and Pro126 that belong to what was called "bonded loop". It should be emphasized that this is the first GAPDH from the phylum Percolozoa that has its three dimensional structure determined and kinetic parameters established, such that we expect to have contributed to the understanding of the evolution of this class of proteins. / O estudo da estrutura tridimensional de proteínas nos permite investigar as relações entre sequência de aminoácidos, estrutura e função, o que é importante principalmente para proteínas de organismos patogênicos ou mesmo pertencente ao gênero destes, que podem ser utilizadas como modelo estrutural. Neste contexto, o presente trabalho visa caracterizar estruturalmente as enzimas alanina racemase isoforma longa de Trypanosoma cruzi e gliceraldeído-3-fosfato desidrogenase de Naegleria gruberi. A alanina racemase isoforma longa catalisa a conversão entre L e D-alanina em uma das vias metabólicas do T. cruzi, que é o agente etiológico da doença de Chagas. A análise por SDS-PAGE de amostras da expressão heteróloga dessa enzima em Escherichia coli BL21(DE3) GroEL revelou que a proteína está em maior proporção na fração insolúvel, por isso, foi necessário estabelecer um protocolo de recuperação seguido de um reenovelamento in vitro. Dados de ensaios enzimáticos e dicroísmo circular revelaram o sucesso do protocolo de recuperação/reenovelamento, o que poderá no futuro contribuir para a busca de inibidores específicos. A gliceraldeído-3-fosfato desidrogenase de Naegleria gruberi catalisa a sexta etapa da via glicolítica do organismo. A enzima NgGAPDH foi expressa em E. coli (DE3) usando-se o vetor pET-15b e então purificada em três passos de cromatografia, dois por afinidade a níquel e um por exclusão por tamanho. A caracterização enzimática foi realizada com a enzima sem a ―his-tag‖; a NgGAPDH apresentou maior atividade em pH 8,0, 25 °C e 10 mM de arsenato, e cooperatividade positiva frente aos substratos G3P e NAD+. Cristais de NgGAPDH sem a ―his-tag‖ apareceram em 3 dias após montagem das gotas e o melhor difratou a 1,94 A de resolução, pertencendo ao grupo espacial P21 com parâmetros de cela a = 83,74 Å, b = 94,55 A, c = 90,93 A e = 99,96 °. A estrutura final refinada apresenta R = 0,1652 e Rfree = 0,2029. O domínio catalítico formado pelos resíduos 134 a 313 é altamente conservado, como esperado, com exceção da Asn145, presente somente em NgGAPDH, enquanto que as demais GAPDHs apresentam Ser ou Thr na posição correspondente. Análises por dinâmica molecular revelaram que a Asn145 tem correlação de movimento com os resíduos Ala123, Thr125 e Pro126, pertencentes ao que se chamou de ―bonded loop‖. Ressalte-se que esta é a primeira GAPDH do filo Percolozoa que tem sua estrutura tridimensional determinada e parâmetros cinéticos estabelecidos, tal que se espera contribuir para o entendimento da evolução dessa classe de proteínas.