Browsing by Author "Chanique, Andrea M."

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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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    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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    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.