Computational design of oxidoreductases for industrial applications

Abstract

Enzyme catalysis has been scaled up for several industrial sectors during the last decades, including pharmaceutics, food and beverages. This raised the interest of other industries such as energy or paper and pulp sectors, for which biocatalysts need to be improved in order to be economically competitive. Molecular simulations play a key role in finding new applications for enzymes and reducing the costs of experimental work. In this thesis, computational techniques have been developed and applied on oxidoreductases for industrial needs, under the framework of the INDOX project. More specifically, investigations have been focused on lignin degradation and valorization by means of flavoproteins and laccases. Flavoproteins are oxidoreductases containing FAD in most of the cases as a prosthetic group. On the other hand, laccases are multicopper-containing oxidoreductases that can oxidize a large variety of substrates. In both cases, oxygen can act as the final electron acceptor, producing hydrogen peroxide and water as by-products respectively, which makes them suitable for green chemistry applications.

Research on flavoproteins - Efforts were made to improve catalytic activity towards 5-hydroxymethylfurfural oxidation and secondary benzyl alcohols. To do so, a Monte Carlo based in-house algorithm was employed to sample the protein-ligand conformational space, revealing two interesting positions for the aryl-alcohol oxidase flavoprotein, 500 and 501. Experimental collaborators found several variants from these positions that improved the activity of this enzyme for secondary benzyl alcohols, which were then computationally characterized with the same methodology. Investigation of the 5-hydroxymethylfurfural biochemical reaction was also performed, revealing that the negative nature of the carboxylic group limits the diffusion of one of the subproducts. This problem was proven to be solved by using another flavoprotein, 5-hydroxymethylfurfural oxidase, whose larger active site allows the diffusion of the negatively charged substrate although with very low activity. The activity of this enzyme increases with the addition of V367/W466F double mutations, and the main reasons have been identified.

Research on laccases - The addition of a ruthenium photosensitizer potentially allows the oxidation of challenging substrates, although the electron back-transfer was presented as the main limitation. Electron transfer calculations were performed empirically, instead of quantum calculations, to selectively attach the photosensitizer to the surface of the enzyme, finding that increasing the directionality towards the trinuclear copper cluster was the best strategy.

In conclusion, it has been shown how computational studies are a useful complement to experimental work, for the benefit of industries. In silico applications are shown to be beneficial in both ends of the process. On one side, predictions and designs are made to guide experiments, narrowing the spectra of mutations and reducing costs. After the experiments, simulations still provide insights of the reaction mechanism and information about the role of certain amino acids, to better understand the experimental results and, as a final instance, refine future predictions.

La catàlisi enzimàtica a escala industrial s’ha aplicat a diferents sectors durant dècades, entre les quals s’inclouen farmacèutiques i indústries de beure i menjar. Això ha incrementat l’interès d’altres sectors, com ara empreses energètiques i de processat del paper, per les quals els biocatalitzadors encara necessiten millores per tal de poder ser competitius econòmicament. Les simulacions a nivell molecular juguen un paper important en trobar noves aplicacions per als enzims i reduint els costs de la part experimental. En aquesta tesi s’han desenvolupat i aplicat tècniques computacionals en oxidoreductases per a la indústria, sota el marc del projecte INDOX. Concretament, les investigacions s’han centrat en la degradació de la lignina i l’aprofitament dels productes obtinguts mitjançant flavoproteïnes i lacases. Les flavoproteïnes son oxidoreductases que contenen majoritàriament FAD com a cofactor. L’activitat de l’aril alcohol oxidasa s’ha millorat per a alcohols benzílics secundaris, mantenint la selectivitat per l’enantiòmer S. D’altra banda, les lacases son oxidoreductases que contenen múltiples àtoms de coure i que poden oxidar una gran varietat de substrats. En concret, s’han realitzat mesures de transferència electrònica empíriques per tal d’unir un fotosensibilitzador a la superfície d’una lacasa, amb la idea de maximitzar la transferència electrònica al clúster tri-nuclear de coure. En ambdós enzims, una molècula d’oxigen pot ser l’acceptor final d’electrons, produint peròxid d’hidrogen i aigua respectivament com a subproductes, per la qual cosa se’ls considera adequats per a aplicacions de química sostenible. En aquesta tesi es demostra que els estudis computacionals són un bon complement del treball experimental, on hi surt beneficiada la indústria. Les aplicacions dels mètodes in silico son útils per a dissenyar i fer prediccions per tal de guiar els experiments, reduir el número de possibilitats on fer mutacions i tanmateix, els costs. A més a més, les simulacions ofereixen la possibilitat de donar explicacions a posteriori sobre els efectes dels mutants obtinguts, així com informació sobre el mecanisme de reacció i el rol dels aminoàcids.