Theory has been used to complement experiment in the development of both drugs and delivery systems. Theoretical methods are capable of identifying the molecular basis of drug formulation inadequacies and systematic theoretical studies may suggest fruitful avenues for material modification. This thesis highlights the utility of computer-based theoretical calculations for guiding the design of drug formulations and enhancing material-drug compatibility and stability. Specifically, the present work explores the applications of semi-empirical methods and atomistic molecular dynamics (MD) simulations to enhance the performance of nano-emulsions and polymer micelle formulations for the delivery of hydrophobic drugs. This work includes three separate studies preceded by an introductory summary of available theoretical techniques. The first study evaluates the accuracy and reliability of semi-empirical methods and MD simulations as means to select suitable excipients to formulate the anti-cancer drug docetaxel in an emulsion. Here, simulations accurately predict the rank order of drug solubility in various excipients, suggesting that simulation is useful for library enrichment. In the second study, a drug conjugation approach is used to further improve the stability and solubility of docetaxel in a triglyceride-based nano-emulsion. Here, optimal conjugates are identified with computer-based theoretical calculations and conjugates with formulation-compatible moieties are synthesized. As predicted, the conjugates exhibit enhanced solubility and loading efficiency in a nano-emulsion. The goal of the third study is to rationally design a stable unimolecular star copolymer that, as a unimer, does not disassemble upon the dilution that accompanies intravenous injection. Here, MD simulation is used to systematically investigate the solution properties of differently composed star copolymers. Overall, star copolymers with a hydrophobic PCL core ≤ 2 kDa and hydrophilic PEG blocks approaching 14.6 kDa per arm are predicted to form unimolecular micelles that remain unimeric at high concentrations. The studies presented in this thesis demonstrate that theoretical approaches are useful for fast pre-screening of drug formulation materials and for the development of delivery systems and drug derivatives.

Abstract Enzymes are potentially superior as catalysts for many industrial chemical processes because of their high specificity, selectivity, minimum energy requirement and environmental friendliness. However, many challenges remain in order to exploit fully the potential of industrial enzymes. The qualities which are needed are catalytic proficiency, availability in high quantities, low price, low product inhibition, and high activity and stability under process conditions. Directed evolution and rational design are the most common strategies to produce enzymes with the desired properties. The TIM barrel is the most frequent and most versatile fold among naturally occurring enzymes. In all known TIM barrel enzymes, the catalytically active residues are located at one end of the barrel structure, while residues maintaining the stability of the fold are found on the opposite end of the barrel. This special architecture of the TIM barrel proteins makes it possible to change catalytic activity of the protein without compromising its stability, which is a perfect start for protein engineering studies. In this research project, a monomeric triosephosphate isomerase (TIM) variant with an engineered binding groove (A-TIM) was created by using a rational design approach. The major aims of this work were (i) to find novel binders and (ii) characterize the new, bigger binding groove using X-ray crystallographic methods. These studies have discovered that monomeric A-TIM can bind compounds completely different from the natural substrate. Studies on three different classes of binder molecules are reported: (i) true substrate analogues of wild type TIM, (ii) substrate analogues that have an extended hydrophobic tail, and (iii) more extended, phosphate containing substrate analogues. In addition to this, the A-TIM active site was shown to be competent. In general these studies illustrate the importance of protein crystallography for characterizing the binding properties of enzyme variants being studied in enzyme discovery projects. / Tiivistelmä Entsyymit voivat toimia ylivoimaisina katalyytteinä monissa kemianteollisuuden prosesseissa johtuen niiden hyvästä spesifisyydestä, valikoimiskyvystä, alhaisesta energiantarpeesta ja ympäristöystävällisyydestä. Näistä ominaisuuksista huolimatta entsyymien kaikkien mahdollisuuksien hyödyntämisen esteenä on monia haasteita. Tarvittavia ominaisuuksia ovat katalyyttinen tehokkuus, saatavuus suurina määrinä, alhainen hinta, alhainen tuoteinhibitio sekä korkea aktiivisuus ja stabiilisuus prosessiolosuhteissa. TIM-tynnyrirakenne on yleisin ja monipuolisin proteiinien laskostumisrakenne luonnossa esiintyvissä entsyymeissä. Tässä rakenteessa katalyyttisesti aktiiviset aminohappotähteet ovat sijoittuneet tynnyrirakenteen toiselle puolelle, kun taas stabiilisuuden kannalta tärkeät aminohappotähteet ovat sijoittuneet kokonaan toiselle puolelle. Tämä erityinen rakenne antaa mahdollisuuden muokata proteiinin katalyyttistä aktiivisuutta vaikuttamatta haitallisesti sen stabiilisuuteen. Tämä on täydellinen lähtökohta proteiininmuokkaukselle. Tässä tutkimusprojektissa käytettiin ns. järkiperäistä suunnittelua monomeerisen trioosifosfaatti-isomeraasivariantin (A-TIM) luomisessa. Tämän tutkimustyön pääasialliset tavoitteet olivat (i) uusien sitoutujien löytäminen ja (ii) uuden, suuremman sitoutumistaskun ominaisuuksien määrittäminen röntgenkristallografisilla menetelmillä. Tässä tutkimuksessa havaittiin, että A-TIM kykenee sitomaan yhdisteitä, jotka ovat täysin erilaisia luonnolliseen substraattiin verrattuna. Tässä tutkimuksessa kuvaillaan kolmenlaisia sitoutujia: (i) todelliset villityypin entsyymin substraattianalogit, (ii) substraattianalogit, joihin on liitetty hydrofobinen hiilivetyketju ja (iii) villityypin substraattia suuremmat sokerifosfaatit. Tämän lisäksi A-TIM:n aktiivisen keskuksen todistettiin olevan toimintakykyinen. Yleisellä tasolla tämä tutkimus osoittaa röntgenkristallografisten menetelmien tärkeyden entsyymienmuokkausprojekteissa, joissa entsyymivarianttien ominaisuuksien määritys on tärkeää.

Les micro-organismes ont la capacité de s'adapter rapidement aux différentes contraintes environnementales ou métaboliques, mais le mécanisme détaillé et les principes de cette réponse adaptative en micro-organisme sont mal compris aux niveaux génétiques, biochimiques et métaboliques. Ici, une souche de E. coli a évolué avec un titre élevé de 1,3-propanediol a été sélectionné et cette souche a été analysée comme un exemple pour la découverte de ce processus d'évolution adaptative. La technologie de séquençage comparatif du génome entier a été utilisée pour identifier les mutations génétiques dans le chromosome et le plasmide portant les gènes codant pour la voie de production de 1,3-propanediol a également été séquencée. Quatre mutations ont été trouvées dans le chromosome et ils sont Ipd, glpR, dhak, nagD - promoteur. Une mutation a été trouvée dans le gène GGP2, qui est situé dans le plasmide. Toutes les mutations ont été en outre caractérisées par analyse génétique et inverse biochimique et leur influence dans le réseau métabolique sont aussi découverts. Nous avons démontré que tous les cinq mutations individuellement peuvent augmenter le taux de croissance et la production de 1,3 -propanediol. Selon le profil de fermentation de la souche évolué, l’accumulation d'acétate entravé la croissance de la souche et de l'augmentation de 1,3-propanediol titre. Pour optimiser la production de 1,3- propanediol, un nouveau catalyseur a été développé pour la co-production de 1,3-propanediol avec de l'acétone au lieu de l'acétate, parce que l'acétone a une plus grande valeur et est moins toxique que l'acétate. Les deux voies de l'acétate, dépendantes et indépendantes, ont été testées pour la co-production de 1,3-propanediol et l'acétone dans des conditions anaérobies. Pour la voie de l'acétate dépendante, en modifiant le niveau d'expression de l'acétate kinase cette voie était active dans des conditions anaérobies dans E. coli. Pour la voie de l'acétate indépendant, une courte chaîne acyl-CoA thioestérase candidat a été sélectionné et caractérisé, mais son activité doit encore être améliorée en utilisant l'évolution adaptative ou la sélection in vitro. Pour l'évolution adaptative, le réseau métabolique était rationnel conçu pour forcer de flux de carbone à la production d'acétone, ce qui va augmenter la possibilité et l'efficacité de la sélection d'une thioestérase avec une affinité et une activité élevée à l'acétoacétyl -CoA au cours du processus d’évolution adaptative. Pour la sélection in vitro, un système de bio-sensor a été développé afin de simplifier la méthode de sélection d'une thioestérase mutant avec une grande capacité de catalyser l'acétoacétyl -CoA in vitro. / Microorganisms have the ability to adapt rapidly to different environmental or metabolic constraints, but the detailed mechanism and the principles of this adaptive response in microorganism is poorly understood at the genetic, biochemical, and metabolic levels. Here, the glycerol pathway from S. cerevisiae and the B12-independent C. butyricum 1,3-PD pathways were introduced into E. coli and its central metabolic network was restructured to couple the production of 1.3-propanediol to the growth of the microorganism. This strain was grown in conditions favouring adaptive evolution for around 1000 hours. An evolved population was selected under optimal conditions in mineral medium. Comparing with the original strain, it can convert glucose to 1.3-PD at high molar yield (94 %) and its productivity was also significantly increased. Comparative whole genome sequencing technology was used to identify the genetic mutations and five mutating genes lpd, glpR, dhaK, nagD and GPP2 were discovered. All the mutations were further analysed and characterized to disclose their changes after the evolution and to elucidate their influence in the whole metabolic engineering network. To optimize the production of 1.3-PD further, we plan to convert the co-production of acetate to acetone. Indeed, the 1.3 propanediol production was hampered by the acetate inhibition on growth, and acetone is a valuable product which is less toxic thyan acetate. Both the acetate-dependent and independent pathways were tested to produce acetone and some modifications to adapt the global metabolic network were performed. Several strategies were applied to ameliorate the performance of acetone production. Finally, the bottleneck of the acetate-dependent acetone pathway under anaerobic condition was indentify and the acetate-independent acetone pathway still need to be improve with the selection of an evolved or mutant enzyme with high short-chain acyl-CoA thioesterase activity.

La Caspase-2 (CASP-2) est singulière de par ses multiples rôles physiologiques et par son implication dans les processus neurodégénératifs aigus et chroniques. Dans ce contexte, des études récentes ont contribué à sa validation en tant que cible thérapeutique potentielle de la maladie d’Alzheimer. Par conséquent, le développement d’inhibiteurs spécifiques constituerait des outils pharmacologiques qui permettraient de mieux appréhender ses rôles dans la physiologie et pathologie du neurone. Les inhibiteurs de Caspases actuels sont majoritairement des séquences tétra ou pentapeptidiques qui reproduisent les motifs préférentiellement reconnus par ces enzymes. Dans le cadre de ce travail de thèse, trois stratégies d’identification d’inhibiteurs ont été suivies ; (i) une approche de conception rationnelle de peptides ciblant le site actif, (ii) conception in silico de peptides de l’interface de dimérisation, (iii) criblage aléatoire et rationnel de petites molécules organiques. Parmi ces stratégies, l’inhibition du site actif s’est révélée être la plus fructueuse. Nous avons ainsi pu démontrer que des variations du résidu Alanine en P2 sur un motif VDVAD permettaient d’améliorer les paramètres de sélectivité et d’efficacité. Sur ce constat une série de peptides « LJ » avec des mécanismes d’inhibition variés a été développée. Deux composés LJ2 et LJ3, ont démontré d’excellents paramètres d’inactivation et de sélectivité envers la CASP-2. Dans des réseaux de neurones reconstruits in vitro, LJ2 et LJ3 présentent un effet synaptoprotecteur. Ces travaux de thèse ouvrent donc le champ à de nouvelles perspectives sur le plan fonctionnel ainsi que sur le plan thérapeutique. / Caspase-2 (CASP-2) is unique among Caspases since its involved in a plethora of physiological processes and in severe and chronic neurodegenerative processes. In this context, recent studies have indicated that CASP-2 is a potential therapeutic target for Alzheimer’s disease. It is therefore necessary to develop specific inhibitors which would constitute pharmacological tools to better understand the role of this protease in the physiology and pathology of the neuron. The current Caspases inhibitors are mostly tetra or pentapeptide sequences that reproduce the patterns preferentially recognized by these enzymes. During this thesis project, we used three identification strategies ; (i) a rational design approach of peptides targeting the active site, (ii) in silico design of peptides of the dimerization interface, (iii) rational an random screening of small organic molecules. Among these strategies, inhibition of the active site has been shown to be the most productive one. We have been able to demonstrate that the variation of the Alanine residue in P2 on the pattern VDVAD increased efficiency and selectivity parameters. Based on this observation, a serie of peptides “LJ” with various inhibitory mechanisms has been developed. Two compounds LJ2 and LJ3, demonstrated excellent inactivation and selectivity parameters toward CASP-2. In neuronal networks reconstructed in vitro, LJ2 and LJ3 protect against synapse loss. This thesis project opens the field to new perspectives on the functional level as well as on the therapeutic plan.

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Made available in DSpace on 2017-07-24T19:38:06Z (GMT). No. of bitstreams: 1 Cibeli May Villalba.pdf: 4939252 bytes, checksum: 5a6b5eaea0352844ad9868a7a9b79f0b (MD5) Previous issue date: 2012-03-13 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Proteins are the most abundant biomolecules in living organisms; they are present in all parts of a cell. They have different functions; their structural study is important because it brings greater insight into its functions and allows us to understand how they interact to each other and with the other molecules. Protein structures can be studied experimentally especially by the X-ray diffraction technique and computationally by homology modeling. Thus, in this work, structural studies were made with the alpha-amylase inhibitor from rye (Secale cereale), which inhibits the activity of amylase and then can be used in the treatment of Diabetes mellitus, obesity, pest control, amongst other applications. Through chromatographies, two different inhibitors could be separated, namely A2 and B2, which were crystallized, but did not show a minimum X-ray diffraction quality. Thus, a structural study was performed with data from a twinned crystal previously obtained, yet current refinement programs can now deal with such data. The structure was refined and compared with the alpha-amylase inhibitor 0.19 from wheat. Then, structural studies were also performed for the YchF GTPase and enolase from Trypanosoma cruzi; both have been studied with the possibility of being used as a target in the treatment of Chagas' disease. Initially, trials to express heterologously and to purify them for crystallization trials were performed; yet those were unsuccessful, a computational work was pursued, in which alignments and homology modelling for both proteins were made. The computational work was continued for Trypanosoma cruzi enolase,in which comparisons with the Homo sapiens enolase to seek and plan inhibitors for the former, through literature and data bank searches, were made; thus, docking of these was performed, which pointed more favorable binding energies for the substrates, phosphoenolpyruvate (PEP) and 2-phosphoglycerate (PG2), for the inhibitor phosphonacetohydroxamate (PAH) and for the compound coded ZINC25695689 from the ZINC (ZINC Is Not Commercial) data bank. Also, from the experimental position of the PAH inhibitor (deposited in the PDB, code 2PTZ), the interaction energies for these searched and planned molecules were estimated,through the AMBER molecular dynamics program, and, apparently, the presence of a chlorine atom conveniently bound to the inhibitor could promote an improvement of the interaction energy. / As proteínas são as biomoléculas mais abundantes nos seres vivos, estando presentes em todas as partes de uma célula. Elas possuem diferentes funções no organismo; seu estudo estrutural é importante, pois traz maior conhecimento sobre suas funções e possibilita entender como interagem entre elas e com outras moléculas. A estrutura de proteínas pode ser estudada experimentalmente principalmente por meio da técnica de difração de raios X e computacionalmente por meio da modelagem por homologia. Sendo assim, realizaram-se neste trabalho estudos estruturais com o inibidor de alfa-amilase do centeio (Secale cereale), que inibem a atividade amilásica e que podem ser utilizados em tratamento de Diabetes mellitus, obesidade, controle de pragas, entre outros usos. Por meio de cromatografias, puderam-se separar dois diferentes inibidores, denominados A2 e B2, que foram cristalizados, mas não apresentaram mínima qualidade de difração de raios X. Assim, realizou-se um estudo estrutural com dados de difração obtidos anteriormente para um cristal geminado, dada a possibilidade atual dos programas de refinamento de tratarem este problema. A estrutura foi refinada e comparada com o inibidor de alfa-amilase 0,19 do trigo. Em sequência, estudos estruturais também foram realizados para a proteína YchF da família das GTPases e para a enolase de Trypanosoma cruzi; ambas vêm sendo estudadas com a possibilidade de serem usadas como alvo no tratamento da doença de Chagas. Inicialmente tentou-se expressá-las de maneira heteróloga e purificá-las para a realização de ensaios de cristalização; com o insucesso disto, partiu-se para um trabalho computacional em que se fizeram alinhamentos e modelos por homologia para as duas proteínas. O trabalho computacional foi continuado para a enolase de Trypanosoma cruzi, comparando-a com a enolase de Homo sapiens para se buscar e planejar inibidores para a primeira, por meio de pesquisa na literatura e em bancos de dados; assim, fez-se a alocação ("docking") destes, obtendo-se energias de ligação mais favoráveis para os substratos, fosfoenolpiruvato (PEP) e 2-fosfoglicerato (PG2), para o inibidor fosfonacetohidroxamato (PAH) e para o composto codificado ZINC25695689 do banco de dados ZINC (ZINC Is Not Commercial). Também, a partir da posição experimental do inibidor PAH (depositada no PDB, código 2PTZ) estimaram-se as energias de interação para as moléculas pesquisadas e planejadas, através do programa de dinâmica molecular AMBER e, aparentemente, a presença de um átomo de cloro convenientemente ligado ao inibidor poderia promover melhoria da energia de interação.

Made available in DSpace on 2017-07-24T19:38:12Z (GMT). No. of bitstreams: 1 VivianePaula.pdf: 3188050 bytes, checksum: 1b1ca9983470b6e6a3669cfd52a8c846 (MD5) Previous issue date: 2007-03-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The enzymes dihydrofolate reductase-thymidylate synthase (DHFR-TS) and pteridine reductase (PTR) are involved in the pterin/folate dependent metabolism; together they represent an important target for chemotherapy of parasitic leishmanias and trypanosomes. Xray crystallography was used to elucidate accurately the structure of the PTR1 enzyme from Trypanosoma brucei in complex with inhibitors which are analogous to the substrate. The ligands assayed for crystallization were the substrate folate and the inhibitors melamine, 6-thioguanine, WSG1012, WSG1034, WSG3065, WSG3066 and WSG3067. Of these, four yielded crystals with diffraction patterns sufficient for a complete dataset. WSG3065 (later revealing the lack of the ligand), WSG3066 and WSG3067 are three of the several structures presented in this work which came from the cited crystallization assays; added to these are the refined structures complexed with triamterene and cyromazine, proceeded from two other datasets already available. The datasets were processed with the programs Mosflm / Scala and Xds / Xscale, the structures were refined using the programs CNS and Refmac5 and validated with the programs Procheck, Whatcheck, Sfcheck and ValidationPDB. All refined structures belong to the space group P21 with unit cells around a = 79, b = 90, c = 82, b = 115, 4 monomers each of 268 residues per asymmetric unit and complex active sites. Besides the inhibiting ligands (except WSG3065) present in the structure, other ligands were found either near or outside the active site: dithiothreitol, glycerol, ethylene glycol, sodium and acetate ions. Analyses on the ligand positions and corresponding interactions with the protein were carried out to understand modes of inhibition and to guide the design or the discovery of new compounds which are potent, but selective to the parasitic enzyme, inhibitors. Thereby, initial docking studies were performed aiming at identifying new molecules or lead compounds with inhibitory capabilities. / As enzimas dihidrofolato redutase-timidilato sintase (DHFR-TS) e pteridina redutase (PTR) estão envolvidas no metabolismo pterina/folato dependente; juntas, representam um importante alvo para a quimioterapia de leishmanias e tripanossomas parasitas. A Cristalografia por Raios X foi utilizada para elucidar acuradamente a estrutura da enzima PTR1 de Trypanosoma brucei complexada com inibidores que são análogos ao substrato. Os ligantes ensaiados para cristalização foram o substrato folato e os inibidores melamina, 6-tioguanina, WSG1012, WSG1034, WSG3065, WSG3066 e WSG3067. Destes, quatro forneceram cristais com padrões de difração suficientes para um conjunto de dados completo. WSG3065 (mais tarde revelando ausência do ligante), WSG3066 e WSG3067 são três das estruturas apresentadas neste trabalho derivadas dos ensaios de cristalização citados; somadas a estas estão as estruturas refinadas dos complexos com triantereno e ciromazina, provenientes de dois outros conjuntos de dados anteriormente disponíveis. Os conjuntos de dados foram processados com os programas Mosflm / Scala e Xds / Xscale, as estruturas refinadas usando-se os programas CNS e Refmac5 e validadas com os programas Procheck, Whatcheck, Sfcheck e ValidationPDB. Todas as estruturas refinadas apresentaram grupo espacial P21 com celas unitárias aproximadas a = 79 = 90, c = 82 , b = 115, 4 monômeros de 268 resíduos cada por unidade assimétrica e sítios ativos complexos. Além dos ligantes inibidores presentes nas estruturas (exceto WSG3065), outros ligantes foram encontrados próximos ou fora do sítio ativo: ditiotreitol, glicerol, etilenoglicol, íons sódio e íons acetato. Análises das posições dos ligantes inibidores e correspondentes interações com a proteína foram realizadas a fim de se entender modos de inibição e, em particular, assistir ao planejamento ou à descoberta de novos compostos que sejam inibidores potentes, mas seletivos, para a enzima parasitária. Assim, estudos iniciais de atracagem (docking) foram realizados visando identificar novas moléculas ou arcabouços com capacidades inibitórias.

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease that leads to degradation of cartilage and bone mainly in peripheral joints. In collagen-induced arthritis (CIA), a mouse model for RA, activation of autoimmune CD4+ T cells depends on a molecular recognition system where T-cell receptors (TCRs) recognize a complex between the class II MHC Aq protein and CII259-273, a glycopeptide epitope from type II collagen (CII). Interestingly, vaccination with the Aq/CII259-273 complex can relieve symptoms and cause disease regression in mice. This thesis describes the use of modified glycopeptides to explore interactions important for binding to the Aq protein and recognition by autoimmune T-cell hybridomas obtained from mice with CIA. The CII259-273 glycopeptide was modified by replacement of backbone amides with different amide bond isosteres, as well as substitution of two residues that anchor the glycopeptide in prominent pockets in the Aq binding site. A three-dimensional structure of the Aq/glycopeptide complex was modeled to provide a structural basis for interpretation of the modified glycopeptide’s immunological activities. Overall, it was found that the amide bond isosteres affected Aq binding more than could be explained by the static model of the Aq/glycopeptide complex. Molecular dynamics (MD) simulations, however, revealed that the introduced amide bond isosteres substantially altered the hydrogen-bonding network formed between the N-terminal 259-265 backbone sequence of CII259-273 and Aq. These results indicated that the N-terminal hydrogen-bonding interactions follow a cooperative model, where the strength and presence of individual hydrogen bonds depended on the neighboring interactions. The two important anchor residues Ile260 and Phe263 were investigated using a designed library of CII259-273 based glycopeptides with substitutions by different (non-)natural amino acids at positions 260 and 263. Evaluation of binding to the Aq protein showed that there was scope for improvement in position 263 while Ile was preferred in position 260. The obtained SAR understanding provided a valuable basis for future development of modified glycopeptides with improved Aq binding. Furthermore, the modified glycopeptides elicited varying T-cell responses that generally could be correlated to their ability to bind to Aq. However, in several cases, there was a lack of correlation between Aq binding and T-cell recognition, which indicated that the interactions with the TCRs were determined by other factors, such as presentation of altered epitopes and changes in the kinetics of the TCR’s interaction with the Aq/glycopeptide complex. Several of the modified glycopeptides were also found to bind well to the human RA-associated DR4 protein and elicit strong responses with T-cell hybridomas obtained from transgenic mice expressing DR4 and the human CD4 co-receptor. This encourages future investigations of modified glycopeptides that can be used to further probe the MHC/glycopeptide/TCR recognition system and that also constitute potential therapeutic vaccines for treatment of RA. As a step towards this goal, three modified glycopeptides presented in this thesis have been identified as candidates for vaccination studies using the CIA mouse model.

Directed evolution is generally regarded as a useful approach in protein engineering. By subjecting members of a mutant library to the power of Darwinian evolution, desired protein properties are obtained. Numerous reports have appeared in the literature showing the success of tailoring proteins for various applications by this method. Is it a one-way track that protein practitioners can only learn from nature to enable more efficient protein engineering? A structure-and-mechanism-based approach, supplemented with the use of reduced amino acid alphabets, was proposed as a general means for semi-rational enzyme engineering. Using human GST A2-2*E, the most active human enzyme in the bioactivation of azathioprine, as a parental enzyme to test this approach, a L107G/L108D/F222H triple-point mutant of GST A2-2*E (thereafter designated as GDH) was discovered with 70-fold increased activity, approaching the upper limit of specific activity of the GST scaffold. The approach was further experimentally verified to be more successful than intuitively choosing active-site residues in proximity to the bound substrate for the improvement of enzyme performance. By constructing all intermediates along all putative mutational paths leading from GST A2-2*E to mutant GDH and assaying them with nine alternative substrates, the fitness landscapes were found to be “rugged” in differential fashions in substrate-activity space. The multidimensional fitness landscapes stemming from functional promiscuity can lead to alternative outcomes with enzymes optimized for other features than the selectable markers that were relevant at the origin of the evolutionary process. The results in this thesis suggest that in this manner an evolutionary response to changing environmental conditions can readily be mounted. In summary, the thesis demonstrates the attractive features of the structure-and-mechanism-based semi-rational directed evolution approach for optimizing enzyme performance. Moreover, the results gained from the studies show that laboratory evolution may refine our understanding of evolutionary process in nature.