Nature employs complex networks of protein-tailoring enzymes to effect the post-translational modification of proteins in vivo. By comparison, modern chemical methods rely upon either nonspecific labeling techniques or upon the genetic incorporation of bioorthogonal handles. To develop truly robust bioconjugates it is necessary to develop methods which possess the exquisite activity and specificity observed in biological catalysts. One attractive strategy to achieve this is the engineering of protein-tailoring enzymes possessing user-defined specificity and high catalytic efficiency. / Chemistry and Chemical Biology

Biocatalysts are increasingly prevalent in the large-scale synthesis of enantiomerically pure compounds. However, many sought-after reactions lack a suitable enzymatic production route. This work describes the development of a novel amine dehydrogenase through the application of directed evolution altering the substrate specificity of an existing leucine dehydrogenase scaffold. Eleven rounds of directed evolution completely altered the enzyme’s specificity and successfully created amination activity. The resulting amine dehydrogenase asymmetrically catalyzes methyl isobutyl ketone and free ammonia to 1, 3-dimethyl butyl amine. The enantioselectivity of the wild-type enzyme was maintained despite the drastic changes to the binding pocket and yielded (R)-1,3-DMBA with nearly complete conversion making it an attractive catalyst in the synthesis of chiral amines. This was the first example of a cofactor-dependent amine dehydrogenase capable of selectively synthesizing chiral amines from a prochiral ketone and free ammonia. Additionally, knowledge gained altering the specificity of the leucine dehydrogenase scaffold was applied to an analogous phenylalanine dehydrogenase scaffold allowing for rapid evolution of novel activity. A single mutational library resulted in a second amine dehydrogenase with enhanced activity toward significantly different substrates, while maintaining comparable conversion and enantioselectivity. These two scaffolds provide examples of the broad applicability of the identified mutations in creating amine dehydrogenase activity.

Glutathione transferases (GSTs) are good candidates for investigations of enzyme evolution, due to their broad substrate specificities and structural homology. The primary role of GSTs is to act as phase II detoxifying enzymes protecting the cell from toxic compounds of both endo- and exogenous origins. The detoxification is conducted via conjugation with glutathione (GSH), which facilitates their removal from the body. The work presented in this thesis has supported a theory for enzyme evolution when the multiple pathway to novel functions can been seen to involve a “generalist” state from which “specialist” states with a new activities can evolve. The generalist has broader specificity and lower activity than the specialist. The term quasi-species is used for a group or cluster of enzyme variants with similar functional properties, and this entity has been suggested as the fittest group for further evolution. This is based on studies of the evolution of new GST variants in two generation. Three diverging clusters or quasi-species, with diverging substrate selectivity, were identified from a GST M1/M2 library, by using directed evolution (family DNA shuffling), multiple substrate screening and multivariate statistics as tools. One of the clusters was M1-like and the other was M2-like, both functionally and structurally. The third quasi-species diverged orthogonally from the parent-like distributions. Its functional character can be referred to as a “generalist” as it had lower activities with most of the substrates assayed except for epoxy-3-(4-nitrophenoxy)-propane (EPNP) and p-nitrophenyl acetate (pNPA). Another round of family DNA shuffling was made with selected variants from the “generalist” quasi-species. From the second generation three quasi-species emerged with diverging functions and sequences. The major cluster contained enzyme variants that represented a direct propagation of the generalists. Diverging from the generalists was a cluster with high specificity with isothiocyanates (ITCs). Increased ITC specificity and decreased epoxide specificity was observed among the novel variants (specialists). The change in functional properties was attributed to a Tyr116His substitution in the active site. These results demonstrate the usefulness of multivariate analysis in the quest for novel enzyme quasi-species in a multi-substrate space, and how minimal changes in the active site can generate distinctive functional properties. An application of our method could be identification of enzyme quasi-species that have lost their sensitivity with alternative inhibitors.

Orientador: Anita Jocelyne Marsaioli / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-19T01:24:15Z (GMT). No. of bitstreams: 1 Mantovani_SimoneMoraes_D.pdf: 4553084 bytes, checksum: 941c2cd131469cbd398d6bc132f763bd (MD5) Previous issue date: 2011 / Resumo: A utilização de biocatalisadores em processos industriais permite a obtenção de produtos de alto valor agregado em sintonia com as demandas de caráter tecnológico e de preservação ambiental. De maneira geral, o desenvolvimento de um biocatalisador envolve inicialmente a triagem da atividade de interesse, seguida da análise das propriedades como seletividade (regio e estereosseletividade) ou estabilidade, que podem ser posteriormente otimizadas por evolução dirigida ou desenho racional até a obtenção de um biocatalisador ótimo. Dessa maneira, os objetivos desse trabalho de tese foram aplicar diferentes metodologias para obtenção de biocatalisadores eficientes, que serão descritos em três capítulos. No capítulo I foi descrita a exploração de novos biocatalisadores por triagem da atividade enzimática em formato miniaturizado de biblioteca metagenômica composta por 864 clones utilizando sondas fluorogênicas, e que levou à detecção de quatro clones ativos para a hidrólise de ésteres. Posteriormente esses clones foram avaliados frente a substratos não modificados permitindo identificar um clone expressando uma enzima de alta quimio e enantiosseletividade para resolução cinética de éster propiônico (E > 100).. No capítulo II foi descrito o mecanismo da desracemização de álcoois secundários por células íntegras de Candida albicans CCT 0776 que consiste em um processo cíclico de oxidação e redução. A primeira etapa é catalisada por uma enzima altamente (S)-seletiva dependente de NADP e O2, seguida de redução pouco seletiva dependente de NADH. Esse sistema foi aplicado para diferentes álcoois e dióis, e possibilitou a detecção dos enantiômeros anti-Prelog com conversões de moderadas a altas (60 a 99 %) e altos excessos enantioméricos (80 a 90%) entre períodos de 20 a 120 h. Por fim, no Capítulo III foi descrito o trabalho de engenharia do citocromo P450Bm3 por exploração combinatória de alanina e posterior evolução dirigida para desmetilação regiosseletiva de substratos volumosos. Essa etapa foi desenvolvida durante o estágio de doutorando em Catech (EUA) sob a supervisão da Profa. Frances Arnold e permitiu a obtenção de variantes capazes de catalisar N-desmetilação de alcalóides e hidroxilação de esteróides com rendimentos moderados (20-80%). Todos esses resultados mostram a variedade de técnicas que podem ser empregadas para o desenvolvimento e aplicações de biocatalisadores visando transformações regio e estereosseletivas eficientes / Abstract: Biocatalysts have been widely applied in recent decades for industrial processes yielding high value products under environmentally friendly reaction conditions. The development of biocatalysts often begins with screening to identify enzymes with suitable activities followed by characterization of the enzymes chemo-, regio- and stereoselectivity or stability. However, identification of new biocatalysts does not always yield enzymes suitable for a given synthetic problem. To overcome this limitation, biocatalysts can be optimized by protein engineering using rational design or directed evolution. In this context, this thesis describes different methodologies that can be explored to obtain efficient biocatalysts for selective transformations. In Chapter I, functional screening of an 864 member metagenomic library derived from soil using a miniaturized assay based on fluorogenic substrates is described. These assays identified four clones capable of ester hydrolysis. Upon further evaluation using high value substrates, one clone, B6, was shown to display high chemo- and enantioselectivity (E>100) for propionic ester hydrolysis. Chapter II describes the study and application of secondary alcohols deracemization using Candida albicans CCT 0776 whole cells. Monitoring the reaction using phenylethanol as a substrate revealed this system furnishes the (R)-enantiomer in high yield and enantiomeric excess mediated by a cyclic process of oxidation and reduction. In summary the first step is catalyzed by a high S- selective enzyme dependent on NADP and O2 followed by a non-selective reduction catalyzed by an NADH-dependent enzyme. This whole cell biocatalyst was applied to different sec-alcohols and diols allowing the detection of the anti-Prelog products in moderate to high conversions (60 and 99%) and high enantiomeric excess (80 and 90%) within 20 to 120 hour incubation times. Finally, in Charpter III the engineering of cytochrome P450Bm3 by alanine combinatorial scanning mutagenesis followed by directed evolution was used to identify enzymes with regioselective demethylation of bulky substrates. These libraries furnished variants capable of catalyzing regioselective N-demethylation of alkaloids and diastereoselective hydroxylation of steroids in moderate yields. Taken together these studies show the variety of techniques that can be applied for development and application of biocatalysts enabling selective and efficient transformations / Doutorado / Quimica Organica / Doutor em Ciências

Thesis advisor: Abhishek Chatterjee / In the last two decades, unnatural amino acid (UAA) mutagenesis has emerged as a powerful new method to probe and engineer protein structure and function. This technology enables precise incorporation of a rapidly expanding repertoire of UAAs into predefined sites of a target protein expressed in living cells. Owing to the small footprint of these genetically encoded UAAs and the large variety of enabling functionalities they offer, this technology has tremendous potential for deciphering the delicate and complex biology of the mammalian cells. We describe the application of this technology to the modification of adeno-associated virus (AAV) for the first time, enabling the generation of vectors with precisely re-engineered cell-targeting for gene therapy. Our UAA-AAV production platform enables the incorporation of UAAs bearing bio-orthogonal reactive handles into multiple specific sites on the virus capsid and their subsequent functionalization with various labeling molecules. Incorporation of an azido-UAA enabled site-specific attachment of a cyclic-RGD peptide onto the capsid, retargeting the virus to the αv β3 integrin receptors, which are overexpressed in tumor vasculature. This work provides a general chemical approach to introduce various receptor binding agents onto the AAV capsid with site selectivity to generate optimized vectors with engineered infectivity. Next, we used our unique UAA-AAV vector as a tool for the directed evolution of more active UAA incorporation machinery in mammalian cells. It is well known that the efficiency of unnatural amino acid mutagenesis in mammalian cells is limited by the suboptimal activity of the suppressor tRNAs currently in use. The ability to improve their performance through directed evolution can address this limitation, but no suitable selection system was previously available to achieve this. We have developed a novel platform for virus-assisted directed evolution of enhanced suppressor tRNAs (VADER) in live mammalian cells. Our system applies selective pressure for tRNA activity via the nonsense suppression-dependent production of UAA-AAV, and selectivity for the specific incorporation of interest comes from a novel virus purification strategy based on the unique chemistry of the UAA. We demonstrated > 10,000-fold selectivity for active tRNAs out of mock libraries and used this system to evolve libraries generated from the commonly used archaeal pyrrolysyl suppressor tRNA, ultimately identifying a variant which is three times as active as the original tRNA. Finally, we used next-generation sequencing to analyze the fate of every library member over the course of the selection and found that our VADER selection scheme is indeed selective for the enrichment of more active tRNA variants. This work provides a general blueprint for the evolution of better orthogonal suppressor tRNAs in mammalian cells. / Thesis (PhD) — Boston College, 2019. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

The creation of artificial enzymes to catalyze desired reactions is a major goal of computational protein design. However, de novo enzymes display low catalytic efficiencies, requiring the introduction of activity-enhancing active site and distal mutations through directed evolution. A better understanding of how mutations introduced by directed evolution contribute to increased enzymatic activity will guide the development of design methods such that efficient enzymes can be designed de novo. Here, we evaluate the structural, functional, and dynamical impacts of active site and distal mutations introduced by directed evolution of the de novo retro-aldolase RA95, an enzyme that presents an important case study in enzyme design due to the significant structural remodelling that was observed during evolution. We observe that the variant RA95-Core, containing only active site mutations introduced by directed evolution, displays activity within one order of magnitude of the fully evolved variant. This suggests that computational enzyme design methods can be improved to create much more efficient enzymes than what was previously achieved in RA95. However, structural changes induced by distal mutations prevent computational recapitulation of the evolved active site on the original design template, indicating that the optimized active site identified through directed evolution could not have been designed de novo using current design methodologies. We suggest strategies for the incorporation of backbone remodelling into design procedures that would allow recapitulation of the evolved retro-aldolase active site, as well as the de novo design of highly efficient enzymes without the need for optimization by directed evolution.

Mycobacterium tuberculosis remains one of the most common worldwide causes of illness and death due to an infectious disease. The emergence of multiple and extreme-drug resistant strains has increased the need to find an effective vaccine for tuberculosis. The goal of our research group is to engineer a temperature-sensitive (TS) M. tuberculosis strain that can be used as a tool in vaccine development. One approach to create TS M. tuberculosis involves the integration of the essential gene ligA encoding a TS NAD+ dependent DNA ligase, which was taken from the psychrophilic organism Pseudoalteromonas haloplanktis. The integration and functioning of ligA was demonstrated in the fast-growing organism Mycobacterium smegmatis. This strain had a TS phenotype with growth limited to below 37°C. The strain was found to have a stable TS phenotype and did not mutate to a temperature-resistant form at a detectable level. Following experiments with the fast growing M. smegmatis, the integration of the ligA gene was attempted in slow-growing M. tuberculosis. Merodiploids of M. tuberculosis containing both the psychrophilic and the WT ligA gene in its chromosome were obtained. The second approach used for the development of TS M. tuberculosis was the directed evolution of native M. tuberculosis essential genes. An advantage of this approach is that the gene encoding the essential protein will resemble the native M. tuberculosis gene and thus will closely match the native transcriptional and translational rates. A system to screen and select for TS essential genes engineered by directed evolution was designed, where the essential gene on the chromosome of E. coli was knocked out and this gene was supplied on a conditionally replicating plasmid. As a first step in developing this directed evolution approach, a family of conditionally replicating plasmids were created and tested in an essential gene knock-out strain of E. coli. / Graduate

Small stable protein domains are attractive scaffolds for engineering affinity proteins due to their high tolerance to mutagenesis without loosing structural integrity. The albuminbinding domain is a 5 kDa three-helix bundle derived from the bacterial receptor Protein G with low-nanomolar affinity to albumin. In this thesis, the albumin-binding domain is explored as a scaffold for engineering novel affinity proteins with the possible benefit of combining a prolonged serum half-life with specific targeting in a single small scaffold protein. Previously, a library was created by randomizing surface-exposed residues in order to engineer affinity to a new target antigen in addition to the inherent albumin affinity. Here, phage display selections were separately performed against the tumor antigens ERBB2 and ERBB3. The ERBB3 selection resulted in a panel of candidates that were found to have varying affinities to ERBB3 in the nanomolar range, while still retaining a high affinity to albumin. Further characterization concluded that the clones also competed for binding to ERBB3 with the natural activating ligand Heregulin. The selections against ERBB2 resulted in sub-nanomolar affinities to ERBB2 where the binding site was found to overlap with the antibody Trastuzumab. The binding sites on ABD to albumin and either target were found in both selections to be mutually exclusive, as increased concentrations of albumin reduced the level of binding to ERBB2 or ERBB3. An affinity-matured ERBB2 binder, denoted ADAPT6, which lacked affinity to albumin was evaluated as a radionuclide-labeled imaging tracer for diagnosing ERBB2-positive tumors. Biodistribution studies in mice showed a high renal uptake consistent with affinity proteins in the same size range and the injected ADAPT quickly localized to the implanted tumor. High contrast images could be generated and ERBB2-expressing tissue could be distinguished from normal tissue with high contrast, demonstrating the feasibility of the scaffold for use as diagnostic tool. In a fourth study, affinity maturation strategies using staphylococcal cell-surface display were evaluated by comparing two replicate selections and varying the stringency. A sub-nanomolar target concentration was concluded to be inappropriate for equilibrium selection as the resulting output was highly variable between replicates. In contrast, equilibrium sorting at higher concentrations followed by kinetic-focused off-rate selection resulted in high output overlap between attempts and a clear correlation between affinity and enrichment. / <p>QC 20160510</p>

Epoxide hydrolase I from Solanum tuberosum (StEH1) and isolated variants thereof has been studied for mapping structure-function relationships with the ultimate goal of being able to in silico predict modifications needed for a certain activity or selectivity. To solve this, directed evoultion using CASTing and an ISM approach was applied to improve selectivity towards either of the enantiomeric product diols from (2,3-epoxypropyl)benzene (1). A set of variants showing a range of activites and selectivities was isolated and characterized to show that both enantio- and regioselectivity was changed thus the enrichment in product purity was not solely due to kinetic resolution but also enantioconvergence. Chosen library residues do also influence selectivity and activity for other structurally similar epoxides styrene oxide (2), trans-2-methyl styrene oxide (3) and trans-stilbene oxide (5), despite these not being selected for.    The isolated hits were used to study varying selectivity and activity with different epoxides. The complex kinetic behaviour observed was combined with X-ray crystallization and QM/MM studies, powerful tools in trying to explain structure-function relationships. Crystal structures were solved for all isolated variants adding accuracy to the EVB calculations and the theoretical models did successfully reproduce experimental data for activities and selectivities in most cases for 2 and 5.  Major findings from calculations were that regioselectivity is not always determined in the alkylation step and for smaller and more flexible epoxides additional binding modes are possible, complicating predictions and the reaction scheme further. Involved residues for the catalytic mechanism were confirmed and a highly conserved histidine was found to have major influence on activity thus suggesting an expansion of the catalytic triad to also include H104. Docking of 1 into the active site of the solved crystal structures was performed in an attempt to rationalize regioselectivity from binding. This was indeed successful and an additional binding mode was identified, involving F33 and F189, both residues targeted for engineering. For biocatalytic purpose the enzyme were was successfully immobilized on alumina oxide membranes to function in a two-step biocatalytic reaction with immobilized alcoholdehydrogenase A from Rhodococcus ruber, producing 2-hydroxyacetophenone from racemic 2.

The use of enzymes as catalysts for chemical transformations has emerged as a “greener” alternative to traditional organic synthesis. An issue to solve though, is that enzymes are designed by nature to catalyze reactions in a living cell and therefore, in many cases, do not meet the requirements of a suitable biocatalyst. By mimicking Darwinian evolution these problems can be addressed in vitro by different types of directed evolution strategies. α-Hydroxy aldehydes and α-hydroxy ketones are important building blocks in the synthesis of natural products, fine chemicals and pharmaceuticals. In this thesis, two alcohol dehydrogenases, FucO and ADH-A, have been studied. Their potentials to serve as useful biocatalysts for the production of these classes of molecules have been investigated, and shown to be good. FucO for its strict regiospecificity towards primary alcohols and that it strongly prefers the S-enantiomer of diol substrates. ADH-A for its regiospecificity towards secondary alcohols, its enantioselectivity and that is has the ability to use a wide variety of bulky substrates. The kinetic mechanisms of these enzymes were investigated using pre-steady state kinetics, product inhibition, kinetic isotope effects and solvent viscosity effects, and in both cases, the rate limiting steps were pin-pointed to conformational changes occurring at the enzyme-nucleotide complex state. These characterizations provide an important foundation for further studies on these two enzymes.   FucO is specialized for activity with small aliphatic substrates but is virtually inactive with aryl-substituted compounds. By the use of iterative saturation mutagenesis, FucO was re-engineered and several enzyme variants active with S-3-phenylpropane-1,2-diol and phenylacetaldehyde were obtained. It was shown that these variants capability to act on larger substrates are mainly due to an enlargement of the active site cavity. Furthermore, several amino acids which are important for catalysis and specificity were identified. Phe254 interacts with aryl-substituted substrates through π-π stacking and may be essential for activity with these larger substrates. One mutation caused a loss in the interactions made between the enzyme and the nucleotide and thereby enhanced the turnover number for the preferred substrate