"Catalytic promiscuous" enzymes, which possess additional activities besides their "native" activity, albeit with a lower efficiency than the main reaction, have become a new frontier for biochemistry and have received considerable attention. Catalytic promiscuity has been suggested to contribute to enzyme evolution through the mechanism of gene duplication followed by specialization of one of the two copies for the new function. Mimicking this evolutionary shortcut could also provide a more efficient route to changing the function of proteins by directed evolution. The promiscuous phosphatase PP1 is a member of the phosphoprotein phosphatase (PPP) gene family, which is critical for the control of many cellular pathways by antagonizing the effects of protein phosphorylation mediated by kinases. The catalytic promiscuity of PP1&gamma WT and two mutants has been investigated with a set of monoanionic and dianioic phosphester substrates. PP1&gamma is an effective catalyst for the hydrolysis of both monoanionic and dianionic phosphate-ester based substrates 1-5, with second-order rate accelerations that fall within the narrow range of 1011 to 1013. While the transition states of the uncatalyzed hydrolysis reactions of these substrates differ, those for the PP1&gamma-catalyzed reactions are similar. Thus, the enzyme catalyzes the hydrolysis of these substrates by transition states that are controlled by the active site environment more than by the intrinsic nature of the substrates. The reason for the inability of PP1&gamma to catalyze the hydrolysis of a sulfate ester is unclear, and unexpected, since the charge and transition state of this substrate are well within the range of those of the phosphorus-based substrates that are effectively catalyzed. Inhibition experiments suggest that the PP1&gamma active site is tolerant of variations in the geometry of bound ligands. This characteristic may permit the effective catalysis even of substrates whose steric requirements may result in perturbations to the positioning of the transferring group, both in the initial enzyme-substrate complex and in the transition state. The conservative mutation of arginine 221 to lysine results in a mutant that more effectively catalyzes monoanionic substrates than the native enzyme. The surprising result in substrate preference from a single, conservative mutation lends support to the notion that mutations following gene duplication can result in an altered enzyme with different catalytic capabilities and preferences, and may, following subsequent mutations, provide a pathway for the evolution of new enzymes.

Phosphoryl transfers are essential chemical reactions in key life processes, including energy production, signal transduction and protein synthesis. They are known for having extremely low reaction rates in aqueous solution, reaching the scale of millions of years. In order to make life possible, enzymes that catalyse phosphoryl transfer, phosphoryl transferases, have evolved to be tremendously proficient catalysts, increasing reaction rates to the millisecond timescale. Due to the nature of the electronic structure of phosphorus atoms, understanding how hydrolysis of phosphate esters occurs is a complex task. Experimental studies on the hydrolysis of phosphate monoesters with acidic leaving groups suggest a concerted mechanism with a loose, metaphosphate-like transition state. Theoretical studies have suggested two possible concerted pathways, either with loose or tight transition state geometries, plus the possibility of a stepwise mechanism with the formation of a phosphorane intermediate. Different pathways were shown to be energetically preferable depending on the acidity of the leaving group. Here we performed computational studies to revisit how this mechanistic shift occurs along a series of aryl phosphate monoesters, suggesting possible factors leading to such change. The fact that distinct pathways can occur in solution could mean that the same is possible for an enzyme active site. We performed simulations on the catalytic activity of β-phosphoglucomutase, suggesting that it is possible for two mechanisms to occur at the same time for the phosphoryl transfer. Curiously, several phosphoryl transferases were shown to be able to catalyse not only phosphate ester hydrolysis, but also the cleavage of other compounds. We modeled the catalytic mechanism of two highly promiscuous members of the alkaline phosphatase superfamily. Our model reproduces key experimental observables and shows that these enzymes are electrostatically flexible, employing the same set of residues to enhance the rates of different reactions, with different electrostatic contributions per residue.

<p>Multifunctional enzymes have alternative functions or activities, known as “moonlighting” or “promiscuous”, which are often hidden behind a native enzyme activity and therefore only visible under special environmental conditions. In this thesis, the active-site of Pseudozyma (formerly Candida) antarctica lipase B was explored for a promiscuous conjugate addition activity. Pseudozyma antarctica lipase B is a lipase industrially used for hydrolysis or transacylation reactions. This enzyme contains a catalytic triad, Ser105-His224-Asp187, where a nucleophilic attack from Ser105 on carboxylic acid/ester substrates cause the formation of an acyl enzyme. For conjugate addition activity in Pseudozyma antarctica lipase B, replacement of Ser105 was assumed necessary to prevent competing hemiacetal formation. However, experiments revealed conjugate addition activity in both wild-type enzyme and the Ser105Ala variant. Enzyme-catalyzed conjugate additions were performed by adding sec-amine, thiols or 1,3-dicarbonyl compounds to various α,β-unsaturated carbonyl compounds in both water or organic solvent. The reactions followed Michaelis-Menten kinetics and the native ping pong bi bi reaction mechanism of Pseudozyma antarctica lipase B for hydrolysis/transacylation was rerouted to a novel ordered bi uni reaction mechanism for conjugate addition (Paper I, II, III). The lipase hydrolysis activity was suppressed more than 1000 times by the replacement of the nucleophilic Ser105 to Ala (Paper III).</p>

In a lipase investigation, Candida antarctica lipase B (CALB) are explored for enzyme catalytic promiscuity. Enzyme catalytic promiscuity is shown by enzymes catalyzing alternative catalytic transformations proceeding via different transition state structures than normal. CALB normally performs hydrolysis reactions by activating and coordinating carboxylic acid/ester substrates in an oxyanion hole prior to nucleophilic attack from an active-site serine resulting in acyl enzyme formation. The idea of utilizing the carbonyl activation oxyanion hole in the active-site of CALB to catalyze promiscuous reactions arose by combining catalytic and structural knowledge about the enzyme with chemical imagination. We choose to explore conjugate addition and direct epoxidation activities in CALB by combining molecular modeling and kinetic experiments. By quantum-chemical calculations, the investigated promiscuous reactions were shown to proceed via ordered reaction mechanisms that differ from the native ping pong bi bi reaction mechanism. The investigated promiscuous activities were shown to take place in the enzyme active-site by various kinetic experiments, but despite this, no enantioselectivity was displayed. The reason for this is unknown, but can be a result of a too voluminous active-site or the lack of covalent coordination of the substrates during enzyme-catalysis (Paper I-IV). Combining enzyme structural knowledge with chemical imagination may provide numerous novel enzyme activities to be discovered. In an ω-transaminase investigation, two (S)-selective ω-transaminases from Arthrobacter citreus (Ac-ωTA) and Chromobacterium violaceum (Cv-ωTA) are explored aiming to improve their catalytic properties. Structural knowledge of these enzymes was provided by homology modeling. A homology structure of Ac-ωTA was successfully applied for rational design resulting in enzyme variants with improved enantioselectivity. Additionally, a single-point mutation reversed the enantiopreference of the enzyme from (S) to (R), which was further shown to be substrate dependent (Paper V). A homology structure of Cv-ωTA guided the creation of an enzyme variant showing reduced isopropyl amine inhibition. / QC20100609

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

Multifunctional enzymes have alternative functions or activities, known as “moonlighting” or “promiscuous”, which are often hidden behind a native enzyme activity and therefore only visible under special environmental conditions. In this thesis, the active-site of Pseudozyma (formerly Candida) antarctica lipase B was explored for a promiscuous conjugate addition activity. Pseudozyma antarctica lipase B is a lipase industrially used for hydrolysis or transacylation reactions. This enzyme contains a catalytic triad, Ser105-His224-Asp187, where a nucleophilic attack from Ser105 on carboxylic acid/ester substrates cause the formation of an acyl enzyme. For conjugate addition activity in Pseudozyma antarctica lipase B, replacement of Ser105 was assumed necessary to prevent competing hemiacetal formation. However, experiments revealed conjugate addition activity in both wild-type enzyme and the Ser105Ala variant. Enzyme-catalyzed conjugate additions were performed by adding sec-amine, thiols or 1,3-dicarbonyl compounds to various α,β-unsaturated carbonyl compounds in both water or organic solvent. The reactions followed Michaelis-Menten kinetics and the native ping pong bi bi reaction mechanism of Pseudozyma antarctica lipase B for hydrolysis/transacylation was rerouted to a novel ordered bi uni reaction mechanism for conjugate addition (Paper I, II, III). The lipase hydrolysis activity was suppressed more than 1000 times by the replacement of the nucleophilic Ser105 to Ala (Paper III).

Combined thermodynamic/kinetic events amount to a kinetically controlled Dynamic Combinatorial Resolution (DCR) process, where the lability of themolecules/aggregates are used to generate dynamics, and the species experiencing the lowest activation energy is selected via kinetic process. Bothinter- and intramolecular processes can be performed using this concept,resulting in complete resolution and associated amplification of the selected species. When intermolecular processes are resolved using this method, an additional advantage is that only a catalytic amount of selector is required tocontrol the system.In this thesis, the Henry and Strecker reactions were developed as efficient C–C bond-forming routes to single and multi-level dynamic covalent systems.These methods efficiently provided a vast variety of substrates from smallnumbers of starting compounds. These dynamic systems, generated underthermodynamic control at mild conditions, were coupled in one-pot processes with kinetically controlled lipase-mediated transacylation. The enzym emediated resolution of the dynamic nitroaldol system led to enantiomericallypure β-nitroacetates in high yield. Furthermore, combination of multi-leveldynamic Strecker systems and lipase-mediated acylation resulted in theresolution of specific α-aminonitriles from the pool.In addition, the asymmetric synthesis of discrete β-nitroalkanol derivatives wassimply achieved, resulting in high yields and high enantiomeric purities through the direct one-pot procedure. Moreover, racemase type activity oflipase enzyme through N-substituted α-aminonitrile structure has been discovered. By use of control experiments together with molecular modeling,the mechanism of the racemization process has been established. Asymmetric synthesis of N-methyl α-aminonitriles was also performed through the dualfunction of lipase, resulting in high yield and good enantio selectivity. / <p>QC 20100818</p>

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-05-04T19:26:38Z No. of bitstreams: 1 lucasfagundesesteves.pdf: 10750065 bytes, checksum: 8871e6f0092a5a329a1cc8099f9a0382 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-05-17T13:33:34Z (GMT) No. of bitstreams: 1 lucasfagundesesteves.pdf: 10750065 bytes, checksum: 8871e6f0092a5a329a1cc8099f9a0382 (MD5) / Made available in DSpace on 2017-05-17T13:33:34Z (GMT). No. of bitstreams: 1 lucasfagundesesteves.pdf: 10750065 bytes, checksum: 8871e6f0092a5a329a1cc8099f9a0382 (MD5) Previous issue date: 2016-02-29 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Esta tese propõe uma investigação teórica do mecanismo de quebra hidrolítica de um modelo para diésteres de fosfato, o íon BDNPP [bis(2,4-dinitrofenil)fosfato], catalisada por um complexo dinuclear de cobre(II) (R1). Esse complexo metálico foi originalmente planejado para mimetizar a estrutura e as propriedades catalíticas do sítio ativo das catecóis oxidases (COs), revelando um caso interessante de promiscuidade catalítica em sistemas biomiméticos. As possibilidades de mecanismo foram cuidadosamente avaliadas através de cálculos de Teoria do Funcional da Densidade (DFT) em fase gás e em fase aquosa com cálculos no ponto dentro do modelo contínuo polarizável (PCM). Dois mecanismos principais foram encontrados. O Mecanismo 1 (Concertado) corresponde a uma reação do tipo SN2 que envolve o ataque da ponte µOH, situada entre os íons Cu(II), ao centro fosfórico da molécula de BDNPP, enquanto que o Mecanismo 2 (Associativo) ocorre através de sucessivas transferências de próton entre o átomo de oxigênio desta mesma ponte com o átomo de oxigênio terminal do grupo fosfato, passando pela formação de um intermediário pentacoordenado estável. O Mecanismo 1 envolve dois possíveis caminhos reacionais para a liberação do íon DNPP [(2,4-dinitrofenil)fosfato] gerado após a quebra hidrolítica. O primeiro caminho reacional (p1) envolve uma transferência de próton – que ocorre entre o átomo de oxigênio que compunha a ponte µ-OH e o átomo de oxigênio terminal do grupo fosfato – imediatamente após a quebra hidrolítica, seguido pela entrada de duas moléculas de água, sendo a etapa de transferência de próton determinante da velocidade. O segundo caminho reacional (p2) envolve a entrada de duas moléculas de água imediatamente após a quebra hidrolítica sem que haja a reação de transferência de próton, sendo a etapa de quebra hidrolítica a etapa determinante da velocidade. Dentre as propostas de mecanismo estudadas o caminho reacional p2 dentro do Mecanismo 1 corresponde ao mais provável, uma vez que possui a menor barreira de reação (ΔG‡ = 23,7 kcal mol-1, em solução aquosa). A constante de velocidade observada experimentalmente, Kobs, vale 1,7 × 10-5 s-1, indicando que o valor calculado teoricamente (K1 = 2.6 × 10-5 s-1) está em excelente acordo com o valor experimental. O efeito isotópico cinético (KIE) foi avaliado para o caminho reacional p2 dentro do Mecanismo 1 com o intuito de entender as alterações estruturais envolvidas na formação do TS1-i (Estado de transição para o Mecanismo 1), caracterizando perfeitamente o mecanismo descrito. O efeito explícito da inclusão de moléculas do solvente foi avaliado de maneira preliminar para apenas uma estrutura do ciclo catalítico para a quebra hidrolítica de ésteres de fosfato, através da utilização do método de Monte Carlo. Os resultados permitem uma análise detalhada da organização das moléculas de solvente ao redor do complexo, podendo servir de ponto de partida para uma análise mais elaborada dos mecanismos reacionais utilizando modelos explícitos para o solvente. O mecanismo de oxidação de catecóis – representado pelo substrato modelo, o 3,5-di-tercbutilcatecol (DTBC) – no sítio ativo do complexo R1 foi avaliado através de cálculos quanto-mecânicos. Embora não tenham sido obtidos resultados conclusivos acerca da cinética da reação, os aspectos estruturais das principais espécies envolvidas no ciclo catalítico foram analisados. / In this thesis the theoretical investigation of the hydrolytic cleavage mechanism of a phosphate diester, BDNPP [bis(2,4-dinitrophenyl)phosphate] in the active site of the dinuclear copper complex, labelled as R1, has been proposed. The metal complex was originally designed to mimic the active site structure as well the catalytic properties of catechol oxidase, revealing an interesting case of catalytic promiscuity in biomimetic systems. The mechanistic possibilities have been carefully evaluated through Density Functional Theory (DFT) calculations in gas phase and in aqueous solution using continuum solvation models with single point calculations within the Polarizable continuum model (PCM). Two reaction mechanisms have been proposed. The Mechanism 1 (Concerted) is a SN2 type mechanism which involves the direct attack of the µ-OH bridge between the two copper(II) ions towards the phosphorus center whereas, the Mechanism 2 (Associative) occurs through two successive proton transferences between the oxygen atom of the bridging hydroxo ligand and another oxygen atom of the phosphate model forming a stable pentacoordinate intermediate. There are two reactions paths for Mechanism 1 to release the DNPP (2,4-dinitrophenylphosphate) ion generated after the hydrolytic cleavage. The first reaction path (p1) involves a proton transfer immediately after the hydrolytic cleavage, being the proton transfer the rate-determining step, followed by the entry of two water molecules. The second reaction path (p2) comprises the entry of two water molecules just after the hydrolytic cleavage without any proton transfer, being the hydrolytic cleavage the rate limiting step. The most probable catalytic path occurs via Mechanism 1, following the second reaction path (p2) once it involves the lowest free energies activation barrier (ΔG‡ = 23.7 kcal mol-1, in aqueous solution). The experimental rate constant, Kobs is 1.7 × 10-5 s-1, indicating that the calculated value, (K1 = 2.6 × 10-5 s-1) is in a very good accordance with the experimental value. Kinetic Isotope Effect (KIE) analysis for the second reaction path (p2) within the Mechanism 1 has also been considered in order to understand the changes taking place in TS1-i (transition state of Mechanism 1) and perfectly characterize the mechanism here described. The solvent effect using explicit water molecules were evaluated in a preliminary fashion for one structure within the catalytic cycle of hydrolytic cleavage of phosphate ester, using the Monte Carlo method. The obtained results allows a detailed analysis of the water molecules organization around the complex, serving as a starting point for an more elaborated study of the reaction mechanisms by using explicit solvent models. The oxidation of catechols – represented herein by the model substrate, 3,5-di-tercbuthylcatechol (DTBC) – in the active site of the R1 complex were evaluated by using quantum-mechanical calculations. The results are not conclusive for the kinetic, but the structural aspects for the main species in the catalytic cycle were studied.