Computational methods for rational screening and engineering of enzyme properties

2010/2011State of the art computational thechniques were applied to several current research toppics in biocatalysis such as substrate promiscuity, reaction promiscuity and high throughput mutant generation and screening. The studied subjects are of great interest to industrial biocatalysis nowadays and can find large application for rational redesign of inefficient biocatalysts and fast substrate engineering and screening. The overall work can be devided into three principal areas, i.e. understanding catalytic mechanisms, description of enzyme-substrate interactions and integration of available computational methods for the development of a novel authomatized tool for enzyme engineering. In each of these areas, the goal has been to test the existing methodologies as well as the development of new descriptors and ready to use strategies.XXIV Ciclo198

Enantioselectivity of Pseudomonas cepacia lipase towards 2-methyl-3(or 4)-arylalkanols: An approach based on the stereoelectronic theory and molecular modeling

For a better understanding of the previously reported enantioselectivity of Pseudomonas cepacia lipase (PCL) in acylation of racemic primary alcohols, 2-methyl-3(or 4)-arylalkanols, molecular modeling of tetrahedral intermediates (TIs) at the active site was performed. The most probable conformers of TIs were elucidated and their interactions with the amino acid residues of the binding pockets at the enzyme active site were studied. The free energy difference between TIs of two enantiomers was approximated by the differences in potential energy and the solvent accesible surface area. Correlation between the HE(His286). . .O gamma (Ser87) hydrogen bond differences of diastereomeric, low energy gauche-TIs, and experimentally determined enantiomeric ratios was found. In agreement with the stereoelectronic theory, the gauche-TI precedes ester release

Computational development of new biocatalyst for plastic degradation through QM/MM methods

Dissertação de mestrado em Biofísica e BionanossistemasOs polímeros sintéticos têm sido produzidos de forma excessiva nestes últimos anos chegando a atingir cerca de 350 milhões de toneladas por ano. A demanda destes plásticos deve-se às suas propriedades, tais como, não degradabilidade, baixo custo, alta resistência, habilidade de serem moldados a pressões e temperaturas altas, entre outros. Contudo, este excesso leva à sua deposição em aterros, que posteriormente são depositados em ambientes aquáticos e terrestres, causando dessa forma consequências adversas para os biossistemas. O método industrial para a degradação de plástico requere pressões e temperaturas altas, e normalmente a adição de solventes orgânicos que levam à formação de poluentes ambientais adicionais. Dessa forma, é necessário uma solução mais viável. A biodegradação de plástico por enzimas biocatalisadoras tem sido estudada e desenvolvida nos últimos anos, sendo a biodegradação de plástico polietileno tereftalato (PET) o mais recorrente. A degradação deste plástico, normalmente resulta nos produtos intermédios tereftalato de bis(2-hidroxietileno) (BHET) e ácido tereftálico mono-(2-hidroxietil) (MHET) e nos produtos finais ácido tereftlálico (TPA) e etileno glicol (EG). Neste projeto, foi estudado o mecanismo catalítico da enzima IsMHETase contra o substrato MHET, através de métodos computacionais, nomeadamente, métodos híbridos de mecânica quântica/mecânica molecular (QM/MM) usando um esquema subtrativo (ONIOM). A parte QM é composta pelos resíduos de aminoácidos essenciais na reação e foi calculada usando a teoria do funcional de densidade, onde o funcional utilizado foi o B3LYP e a base de funções 6-31G(d,p). Entretanto, a parte MM que envolve o resto do sistema foi calculada através de mecânica molecular, nomeadamente por campos de força ff14SB e GAFF. Os resultados revelaram um mecanismo catalítico de cinco passos, que são divididos em acilação e desacilação. Durante o processo de acilação, Ser225 é desprotonada pela His528 e torna-se um nucleófilo que ataca o grupo carbonilo do substrato MHET, originando o intermediário ácil-enzima e libertando o produto EG. Durante a desacilação, uma molécula de água é desprotonada pela His528 que ataca o intermediário, libertado o segundo produto TPA.Synthetic polymers are being produced in an excessive way, reaching about 350 million tons yearly. The demand of these plastics is due to their proprieties, such as non-degradability, low cost, high resistance, ability to be moulded at high pressures and temperatures, many others. However, the excess leads to its deposition in landfills that end up deposited in aquatic and terrestrial environments, causing several hazard consequences to the biosystems. Industrial strategy for plastic degradation requires high pressure and temperature, and often organic solvents, causing additional environmental pollutants. Therefore, it is necessary a more environmentally friendly strategy. Plastic biodegradation by biocatalyst enzymes have been studied and developed in recent years, being the polyethylene terephthalate (PET) the most studied plastic. The degradation of this plastic normally results in bis(2-hydroxyethyl)terephthalic acid (BHET) and mono-(2-hydroxyethyl)terephthalic acid (MHET) intermediates and terephthalic acid (TPA) and ethylene glycol (EG) building blocks. In this project, the catalytic mechanism of IsMHETase enzyme against MHET substrate was studied, using computational means, namely hybrid quantum mechanical/molecular mechanical (QM/MM) methodology with subtractive scheme (ONIOM). The QM part is composed of key amino acid residues involved in the reaction and was calculated using density functional theory with the functional B3LYP and the basis set 6-31G(d,p). Meanwhile, the MM part encompasses the remaining of the system and was calculated using molecular mechanics, namely ff14SB and GAFF force fields. The results showed a five-step catalytic mechanism that are divided in acylation and deacylation. In acylation, Ser225 is deprotonated by His528, becoming a nucleophile and attacks the carbonyl group of MHET substrate, resulting in the formation of the acyl-intermediate enzyme and the release of EG. In deacylation, a water molecular is deprotonated by His528 and attacks the intermediate, resulting in the second product TPA

Computational study and rational design of pluriZymes

[eng] The increase in production over the last centuries has come at the expense of compromising the environment, urging the need to find solutions. Enzymes are the essential molecules that make life kinetically possible. In industry, enzymes can be a sustainable alternative to using inorganic catalysts. However, their low productivity, poor resistance to industrial conditions, and their cost limit their usage. Thus, enabling the tailoring of biocatalysts at will is crucial to expand their application. The advances in computational power, followed by the repertoire of modeling tools, are helping design the next generation of biocatalysts due to their lower costs and quickness. This thesis aims to develop a novel concept of biocatalysis, which could lower the expression costs of enzymes, named pluriZymes. PluriZymes are proteins with plural catalytic active sites where one (at least) of them is artificially designed. The type of introduced functional site along the thesis has been the hydrolase one due to its simplicity (only 3 catalytic residues needed) and does not need a cofactor. The studied systems were transaminases and esterases since they have several applications in industry, thus, being of broad interest. All computational designs were experimentally validated by our collaborators. The thesis' results include an in-one protease-esterase pluriZyme, a transaminase- esterase pluriZyme with potential applications for the pharmaceutical industry, the rational improvement of substrate promiscuity of hydrolase sites, and a new algorithm to facilitate the design of artificial active sites. Hence, this thesis proves the potential of pluriZymes for the next generation of biocatalysts toward a more sustainable society and the need for computational tools to develop them.[cat] L’augment en la producció dels darrers segles s’ha produït a canvi de comprometre el medi ambient, apressant la necessitat de trobar solucions. Els enzims són les molècules essencials que fan la vida possible cinèticament. En l'àmbit industrial, els enzims poden ser una alternativa sostenible a l’ús de catalitzadors inorgànics. No obstant això, la seva baixa productivitat, la poca resistència a les condicions industrials i el seu cost limiten el seu ús. Així doncs, la capacitat de poder adaptar els biocatalitzadors a voluntat és crucial per ampliar la seva aplicació. Els avenços en els recursos computacionals, seguits pel repertori d’eines de modelatge, estan ajudant a dissenyar la propera generació de biocatalitzadors pels seus baixos costs i la seva rapidesa. Aquesta tesi pretén desenvolupar un nou concepte en biocatàlisi, que podria reduir els costs d’expressió dels enzims, anomenat “pluriZyme”. Els “pluriZymes” són proteïnes amb múltiples llocs actius catalítics on almenys un d’ells està dissenyat artificialment. El tipus de lloc funcional introduït al llarg de la tesi ha estat la hidrolasa per la seva simplicitat (només calen 3 residus catalítics) i no necessita cofactor. Els sistemes estudiats van ser transaminases i esterases, ja que tenen diverses aplicacions a la indústria, per tant, són d'ampli interès. Tots els dissenys computacionals van ser validats experimentalment pels nostres col·laboradors. Els resultats de la tesi inclouen un “pluriZyme” proteasa-esterasa tot en un, un “pluriZyme” transaminasa-esterasa amb aplicacions potencials per a la indústria farmacèutica, la millora racional de la promiscuïtat de substrats de llocs hidrolasa i un nou algorisme per facilitar el disseny de llocs actius artificials. Per tant, aquesta tesi demostra el potencial de pluriZymes per a la propera generació de biocatalitzadors cap a una societat més sostenible i la necessitat d'eines computacionals per desenvolupar-los.[spa] El incremento en la producción de los últimos siglos se ha producido a expensas de comprometer el medioambiente, lo que ha acelerado la necesidad de encontrar soluciones. Las enzimas son moléculas esenciales para que la vida sea cinéticamente posible. En el ámbito industrial, las enzimas pueden ser una alternativa sostenible a los catalizadores inorgánicos. Sin embargo, su baja productividad, poca resistencia a las condiciones industriales y su costo limitan su uso. Por lo tanto, permitir la adaptación de biocatalizadores a voluntad es crucial para expandir su aplicación. Los avances en recursos computacionales, seguidos por el repertorio de herramientas de modelado, están ayudando a diseñar la próxima generación de biocatalizadores debido a su menor costo y rapidez. Esta tesis tiene como objetivo desarrollar un concepto novedoso en el campo de biocatálisis, que podría reducir los costes de expresión de las enzimas, denominado "pluriZymes". Los "pluriZymes" son proteínas con sitios activos catalíticos plurales donde al menos uno de ellos está diseñado artificialmente. El tipo de sitio funcional introducido a lo largo de la tesis ha sido el de hidrolasa debido a su sencillez (solo se necesitan 3 residuos catalíticos) y no necesita cofactor. Los sistemas estudiados fueron transaminasas y esterasas, ya que tienen varias aplicaciones en la industria, por lo tanto, son de amplio interés. Todos los diseños computacionales fueron validados experimentalmente por nuestros colaboradores. Los resultados de la tesis incluyen una proteasa- esterasa "pluriZyme" todo en uno, una transaminasa-esterasa "pluriZyme" con aplicaciones potenciales para la industria farmacéutica, la mejora racional de la promiscuidad de sustratos de sitios hidrolasa y un nuevo algoritmo para facilitar el diseño de sitios activos artificiales. Por lo tanto, esta tesis demuestra el potencial de pluriZymes para la próxima generación de biocatalizadores hacia una sociedad más sostenible y la necesidad de herramientas computacionales para desarrollarlos