Produtos naturais constituem uma das principais fontes de moléculas bioativas que possuem diversas aplicabilidades. Dentre os produtos naturais, policetídeos representam uma ampla classe de compostos estruturalmente diversos cuja atividade biológica, muitas vezes está relacionada com os grupos funcionais que estão ligados ao seu esqueleto central (aglicona). Os macrolídeos representam uma classe de antibiótico amplamente utilizado e são um exemplo de policetídeos cuja atividade é dependente de moléculas de açúcares. As enzimas que realizam a glicosilação de policetídeos são as glicosiltransferases, as quais apresentam uma especificidade estrita para 6-desoxiaçúcares, porém uma especificidade relaxada para açúcares não usuais e substratos aceptores. Estudar essa flexibilidade catalítica das glicosiltransferases de produtos naturais pode contribuir para a geração de novos compostos através de glicodiversificação. Esses novos compostos podem apresentar novas atividades biológicas e propriedades farmacocinéticas melhoradas. Além da especificidade relaxada, existe um pequeno grupo de glicosiltransferases que possui um comportamento peculiar no qual uma proteína auxiliar é necessária para sua atividade catalítica, por exemplo o par TylM2/TylM3 envolvidos na biossíntese do antibiótico macrolídeo tilosina em Strepromyces fradiae. Estudar a interação e as mudanças conformacionais que ocorrem durante a formação do complexo glicosiltransferase-proteína auxiliar é fundamental para entender a influência que essas as proteínas auxiliares exercem sobre as glicosiltransferases, e com isso gerar informações que possam ser úteis na aplicação de glicodiversificação. Neste trabalho foi realizado a sublonagem de genes sintéticos que codificam a glicosiltransferase TylM2 e a proteína auxiliar TylM3 e as proteínas recombinantes foram produzidas e purificadas. Além disso foram realizadas técnicas de caracterização estrutural para proteína TylM2 no qual essa glicosiltransferase parece formar um tetrâmero em solução na ausência de sua proteína auxiliar. Ensaios de cristalização com TylM2 rendeu cristais que difratam a uma resolução abaixo de 3,0Å, mesmo após tentativas de otimização, que dificultam a determinação de sua estrutura A criação de modelos teóricos por modelagem comparativa para TylM2 e TylM3 permitiu uma investigação sobre possíveis diferenças entre a TylM2 e suas homólogas. / Natural products compose one of the main sources of bioactive molecules that have several applications. Amongst natural products, polyketides represent a broad class of structurally diverse compounds whose biological activity is often related to the functional groups that are linked to their central skeleton (aglycone). Macrolides represent a class of widely used antibiotic and are an example of polyketides whose activity is dependent on sugar molecules. The enzymes that perform the glycosylation of polyketides are glycosyltransferases, which have a strict specificity for 6-deoxy sugars, but a relaxed specificity for unusual sugars and acceptor substrates. Studying this catalytic flexibility of glycosyltransferases of natural products may contribute to the generation of novel compounds through glycodiversification. These novel compounds may exhibit new biological activities and improved pharmacokinetic properties. In addition to the relaxed specificity, there is a small group of glycosyltransferases who have a peculiar behavior in which an auxiliary protein is required for its catalytic activity, an example of such is the TylM2 / TylM3 pair involved in the biosynthesis of the macrolide antibiotic tylosin in Strepromyces fradiae. Studying the interaction and conformational changes that occur during the formation of the glycosyltransferase-auxiliary protein complex is critical to understanding the influence that these auxiliary proteins have on glycosyltransferases, and thereby generate information that may be useful in the application of glycodiversification. In this work, synthetic genes coding the glycosyltransferase TylM2 and the auxiliary protein TylM3 was sub cloned into pET28a vectors and the recombinant proteins were produced and purified. In addition, structural characterization techniques were performed with TylM2 which appears to form a tetramer in solution in the absence of its auxiliary protein. Crystallization assays of TylM2 yielded crystals that diffracted bellow 3.0Å and presented pathologies which prevented determining of its structure, even after attempts of optimization. The creation of theoretical models by homology modelling for TylM2 and TylM3 allowed for an investigation into possible differences that make TylM2 possess a more stringent flexibility toward acceptor substrates when compared to other homologues.

Oxygenic photosynthesis is the major producer of both oxygen and organic compounds on earth and takes place in plants, green algae and cyanobacteria. The thylakoid membranes are the site of the photosynthetic light reactions that involve the concerted action of four major protein complexes known as photosystem II (PSII), cytochrome b6f complex, ATP synthase and photosystem I (PSI). The function of PSII is of particular interest as it performs the light–driven water splitting reaction driving the photosynthetic electron transport. My thesis addressed different aspects of PSII assembly and the functions of its low molecular weight PSII subunits PsbX and PsbW. Photosynthesis in green algae and higher plants is controlled by the nucleus. Many proteins of nuclear origin participate in the regulation of the efficient assembly of the photosynthetic protein complexes. In this investigation we have identified one of these nuclear encoded auxiliary proteins of photosystem II, REP27, which participates in the assembly of the D1 reaction center protein and repair of photodamaged PSII in the green algae Chlamydomonas reinhardtii. Interestingly, PSII is specially enriched in Low Molecular Weight (LMW) subunits that have masses less than 10kDa. These proteins account for more than the half of the PSII subunits. Several questions remains poorly understood regarding the LMW: Which is their evolutionary origin? What function do they perform in the protein complex? Where are they located in the protein structure? In this investigation the functions of two of these LMW subunits (PsbX and PsbW) have been studied using antisense inhibition and T-DNA knockout mutant plants in Arabidopsis thaliana. Deficiency of the PsbX protein leads to impaired accumulation and functionality of PSII. Characterization of PsbW knock-out plants show that PsbW participates in stabilization of the macro-organization of PSII and the peripheral antenna (Light Harvesting Complex, LHCII) in the grana stacks of the chloroplast, also known as PSII-LHCII supercomplexes.