Browsing by Author "Kourist, Robert"

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    A Cold-Active Flavin-Dependent Monooxygenase from Janthinobacterium svalbardensis Unlocks Applications of Baeyer-Villiger Monooxygenases at Low Temperature
    (2023) Chanique, Andrea M.; Polidori, Nakia; Sovic, Lucija; Kracher, Daniel; Assil-Companioni, Leen; Galuska, Philipp; Parra, Loreto P.; Gruber, Karl; Kourist, Robert
    Cold-active enzymes maintain a large part of their optimal activity at low temperatures. Therefore, they can be used to avoid side reactions and preserve heat sensitive compounds. Baeyer-Villiger monooxygenases (BVMO) utilize molecular oxygen as a co-substrate to catalyze reactions widely employed for steroid, agrochemical, antibiotic, and pheromone production. Oxygen has been described as the rate-limiting factor for some BVMO applications, thereby hindering their efficient utilization. Considering that oxygen solubility in water increases by 40% when the temperature is decreased from 30 to 10 degrees C, we set out to identify and characterize a cold-active BVMO. Using genome mining in the Antarctic organism Janthinobacterium svalbardensis, a cold active type II flavin-dependent monooxygenase (FMO) was discovered. The enzyme shows promiscuity toward NADH and NADPH and high activity between 5 and 25 degrees C. The enzyme catalyzes the monooxygenation and sulfoxidation of a wide range of ketones and thioesters. The high enantioselectivity in the oxidation of norcamphor (eeS = 56%, eeP > 99%, E > 200) demonstrates that the generally higher flexibility observed in the active sites of cold-active enzymes, which compensates for the lower motion at cold temperatures, does not necessarily reduce the selectivity of these enzymes. To gain a better understanding of the unique mechanistic features of type II FMOs, we determined the structure of the dimeric enzyme at 2.5 angstrom resolution. While the unusual N-terminal domain has been related to the catalytic properties of type II FMOs, the structure shows a SnoaL-like N-terminal domain that is not interacting directly with the active site. The active site of the enzyme is accessible only through a tunnel, with Tyr-458, Asp-217, and His-216 as catalytic residues, a combination not observed before in FMOs and BVMOs.
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    A Structural View on the Stereospecificity of Plant Borneol-Type Dehydrogenases
    (2021) Chánique Sallusti, Andrea Magdalena; Dimos, Nicole; Drienovská, Ivana; Calderini, Elia; Pantín, Mónica P.; Helmer, Carl P. O.; Hofer, Michael; Sieber, Volker; Parra, Loreto; Loll, Bernhard; Kourist, Robert
    The development of sustainable processes for the valorization of byproducts and other waste streams remains an ongoing challenge in the field of catalysis. Racemic borneol, isoborneol and camphor are currently produced from α-pinene, a side product from the production of cellulose. The pure enantiomers of these monoterpenoids have numerous applications in cosmetics and act as reagents for asymmetric synthesis, making an enzymatic route for their separation into optically pure enantiomers a desirable goal. Known short-chain borneol-type dehydrogenases (BDHs) from plants and bacteria lack the required specificity, stability or activity for industrial utilization. Prompted by reports on the presence of pure (−)-borneol and (−)-camphor in essential oils from rosemary, we set out to investigate dehydrogenases from the genus Salvia and discovered a dehydrogenase with high specificity (E>120) and high specific activity (>0.02 U mg−1) for borneol and isoborneol. Compared to other specific dehydrogenases, the one reported here shows remarkably higher stability, which was exploited to obtain the first three-dimensional structure of an enantiospecific borneol-type short-chain dehydrogenase. This, together with docking studies, led to the identification of a hydrophobic pocket in the enzyme that plays a crucial role in the stereo discrimination of bornane-type monoterpenoids. The kinetic resolution of borneol and isoborneol can be easily integrated into the existing synthetic route from α-pinene to camphor thereby allowing the facile synthesis of optically pure monoterpenols from an abundant renewable source.
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    CryoEM analysis of small plant biocatalysts at sub-2 Å resolution
    (2022) Dimos, Nicole; Helmer, Carl P. O.; Chanique, Andrea M.; Wahl, Markus C.; Kourist, Robert; Hilal, Tarek; Loll, Bernhard
    Enzyme catalysis has emerged as a key technology for developing efficient, sustainable processes in the chemical, biotechnological and pharmaceutical industries. Plants provide large and diverse pools of biosynthetic enzymes that facilitate complex reactions, such as the formation of intricate terpene carbon skeletons, with exquisite specificity. High-resolution structural analysis of these enzymes is crucial in order to understand their mechanisms and modulate their properties by targeted engineering. Although cryo-electron microscopy (cryoEM) has revolutionized structural biology, its applicability to high-resolution structural analysis of comparatively small enzymes has so far been largely unexplored. Here, it is shown that cryoEM can reveal the structures of plant borneol dehydrogenases of similar to 120 kDa at or below 2 angstrom resolution, paving the way for the rapid development of new biocatalysts that can provide access to bioactive terpenes and terpenoids.
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    Phenotype based discovery of selected oxidoreductases for the utilization of renewable resources
    (2021) Chánique Sallusti, Andrea Magdalena; Parra, Loreto; Kourist, Robert; Pontificia Universidad Católica de Chile. Escuela de Ingeniería
    El descubrimiento de nuevos biocatalizadores que se adapten a diversas condiciones de proceso es fundamental para implementar la biocatálisis a nivel industrial. Una técnica valiosa para buscar nuevas enzimas es el descubrimiento basado en el fenotipo, en el que las características que se encuentran en el hábitat de un organismo se utilizan como un indicador de la presencia de enzimas que funcionan en esas condiciones. El mismo razonamiento puede ser utilizado al encontrar un producto de interés en el ambiente: probablemente los organismos que viven allí producen enzimas capaces de sintetizar y degradar estos compuestos. Posteriormente, se pueden aplicar técnicas de ingeniería de proteínas a las nuevas enzimas y mejorar más aún sus características. En este trabajo, se utilizó el descubrimiento basado en fenotipo para identificar nuevas oxidorreductasas con características de relevancia industrial. La investigación se centró en encontrar nuevos miembros de dos familias: monooxigenasas de Bayer-Villiger (BVMO) y borneol deshidrogenasas de cadena corta (BDH). En el caso de BVMO, la búsqueda en el genoma de microorganismos antárticos condujo al descubrimiento de una monooxigenasa activa en frío. La enzima muestra una temperatura óptima de 20°C, presenta un 70% de su actividad óptima a 5°C y tiene una buena tolerancia a solventes orgánicos. La enzima muestra promiscuidad de cofactor y cataliza la monooxigenación de sustratos como norcamphor y biciclo[3.2.0]hept-2-en-6-ona de manera regio y enantioselectiva. En el caso de la familia de BDH, se realizó una búsqueda en el genoma de plantas que contienen un alto porcentaje de borneol o alcanfor en sus aceites esenciales, lo que condujo al descubrimiento de enzimas capaces de catalizar la oxidación de borneol de forma enantioespecífica. Algunas de estas enzimas también catalizan la reducción de alcanfor de manera diastereoselectiva. La determinación de las estructuras tridimensionales de dos de estas enzimas permitió estudiar los determinantes estructurales de la especificidad y selectividad. Se utilizó comparación de secuencias, comparación de estructuras, acoplamiento molecular y mutagénesis dirigida para dilucidar la influencia de los residuos del sitio activo en estas características. También se utilizó reconstrucción de secuencias ancestrales para estudiar la historia evolutiva de esta familia en términos de especificidad y termoestabilidad. Esto llevó a la caracterización de ancestros con una especificidad moderada, en contraste con las enzimas existentes, que presentan una especificidad muy alta (E>200) o baja.
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    Rational Design of Resveratrol O-methyltransferase for the Production of Pinostilbene
    (2021) Herrera, Daniela P.; Chanique, Andrea M.; Martinez-Marquez, Ascension; Bru-Martinez, Roque; Kourist, Robert; Parra, Loreto P.; Schuller, Andreas
    Pinostilbene is a monomethyl ether analog of the well-known nutraceutical resveratrol. Both compounds have health-promoting properties, but the latter undergoes rapid metabolization and has low bioavailability. O-methylation improves the stability and bioavailability of resveratrol. In plants, these reactions are performed by O-methyltransferases (OMTs). Few efficient OMTs that monomethylate resveratrol to yield pinostilbene have been described so far. Here, we report the engineering of a resveratrol OMT from Vitis vinifera (VvROMT), which has the highest catalytic efficiency in di-methylating resveratrol to yield pterostilbene. In the absence of a crystal structure, we constructed a three-dimensional protein model of VvROMT and identified four critical binding site residues by applying different in silico approaches. We performed point mutations in these positions generating W20A, F24A, F311A, and F318A variants, which greatly reduced resveratrol's enzymatic conversion. Then, we rationally designed eight variants through comparison of the binding site residues with other stilbene OMTs. We successfully modified the native substrate selectivity of VvROMT. Variant L117F/F311W showed the highest conversion to pinostilbene, and variant L117F presented an overall increase in enzymatic activity. Our results suggest that VvROMT has potential for the tailor-made production of stilbenes.