Abstract
Unspecific peroxygenases (UPOs) enable oxyfunctionalizations of a broad substrate range with unparalleled activities. Tailoring these enzymes for chemo- and regioselective transformations represents a grand challenge due to the difficulties in their heterologous productions. Herein, we performed protein engineering in Saccharomyces cerevisiae using the MthUPO from Myceliophthora thermophila. More than 5300 transformants were screened. This protein engineering led to a significant reshaping of the active site as elucidated by computational modelling. The reshaping was responsible for the increased oxyfunctionalization activity, with improved kcat/Km values of up to 16.5-fold for the model substrate 5-nitro-1,3-benzodioxole. Moreover, variants were identified with high chemo- and regioselectivities in the oxyfunctionalization of aromatic and benzylic carbons, respectively. The benzylic hydroxylation was demonstrated to perform with enantioselectivities of up to 95% ee. The proposed evolutionary protocol and rationalization of the enhanced activities and selectivities acquired by MthUPO variants represent a step forward toward the use and implementation of UPOs in biocatalytic synthetic pathways of industrial interest.
Highlights
Fungal unspecific peroxygenases (UPOs) are heme-containing proteins that catalyze oxyfunctionalization reactions of a broad substrate scope via an oxyferryl active species known as compound I (Cpd I), analogous to hemeperoxidases and P450 monooxygenases (P450s).[1−4] UPOs utilize hydrogen peroxide as a “prereduced” oxygen source and do not require additional reducing agents or reductase domains such as P450s, which require NAD(P)H equivalents and electron transfer steps to activate molecular oxygen (Scheme 1).[5−7] This facile Cpd I generation and its high activities render UPOs as very promising biocatalysts
Aromatic oxidations could lead to the respective quinones, such as naphthoquinone from naphthalene, which is assumed to proceed via the 1-naphthol formation followed by a peroxidase-type single electron oxidation.[12,13]
The recently characterized MthUPO was engineered using a protein evolution protocol that involves site saturation mutagenesis and an extensive recombination library, making use of a split-GFP and colorimetric assays and a highthroughput yeast expression system that allowed us to screen more than 5300 transformants
Summary
Fungal unspecific peroxygenases (UPOs) are heme-containing proteins that catalyze oxyfunctionalization reactions of a broad substrate scope via an oxyferryl active species known as compound I (Cpd I), analogous to hemeperoxidases and P450 monooxygenases (P450s).[1−4] UPOs utilize hydrogen peroxide as a “prereduced” oxygen source and do not require additional reducing agents or reductase domains such as P450s, which require NAD(P)H equivalents and electron transfer steps to activate molecular oxygen (Scheme 1).[5−7] This facile Cpd I generation and its high activities render UPOs as very promising biocatalysts. UPOs have demonstrated in the last two decades to be highly efficient biocatalysts for carbon, sulfur, and nitrogen oxyfunctionalizations.[8−11] They can activate C−H bonds of sp3-hybridized carbons enabling a homolytic cleavage. The functionalization of C−C double bonds results in an epoxide formation, while for aromatic oxidations, the initial epoxidation follows a spontaneous rearomatization resulting in a formal hydroxylation product. While the activity and stereoselectivity of UPO-catalyzed reactions are auspicious for future synthetic and industrial applications, low regio- and chemoselectivities mostly afford product mixtures hampering their direct utilization
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