Accessing Chemo- and Regioselective Benzylic and Aromatic Oxidations by Protein Engineering of an Unspecific Peroxygenase

Abstract

Unspecific peroxygenases (UPOs) enable oxyfunctionalisations 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 a protein engineering in <i>S. cerevisiae</i> with the novel <i>Mth</i>UPO. Experimental approaches were combined with computational modelling resulting in the screening of more than 5,300 transformants. This protein engineering led to a significant reshaping of the active site as elucidated by molecular dynamics. The k<sub>cat</sub>/K<sub>m</sub> was improved by 16.5-fold. Variants were identified with high chemo- and regioselectivities in the oxyfunctionalisation of aromatic and benzylic carbons, respectively. The benzylic hydroxylation was demonstrated to perform with excellent enantioselectivities of 95 % <i>ee</i>. Additionally, the first reported effective exchange of the conserved catalytic Glu residue was observed.

Enzymatic Hydroxylations of sp3-Carbons

“…By screening more than 5000 transformants, the catalytic efficiency on the conversion of the model substrate 5-nitro-1,3-benzodioxole (NBD) was improved 16.5-fold. Improved variants led to the chemodivergent formation of selective oxyfunctionalizations at the aromatic 37 and benzylic positions 36 , respectively, using 2-methylnaphthalene ( 35 ) as a substrate (Scheme C) …”

“…Improved variants led to the chemodivergent formation of selective oxyfunctionalizations at the aromatic 37 and benzylic positions 36, respectively, using 2-methylnaphthalene (35) as a substrate (Scheme 10C). 61 CglUPO from Chaetomium globosum can catalyze the epoxidation and hydroxylation of testosterone (38) yielding testosterone 4,5 β-epoxide (40) and 16α-hydroxy testosterone (39) (Scheme 10D). Those steroids exhibit crucial roles in physiological processes.…”

Simultaneous screening of multiple substrates with an unspecific peroxygenase enabled modified alkane and alkene oxyfunctionalisations

“…To gain insights into the switch in the hydroxylation pattern observed due to the A161L mutation, we performed molecular dynamics (MD) simulations as previously shown for UPOs. 28,30 MD simulations with octane substrate bound in wild type MthUPO showed that the preferential binding conformation of octane corresponds to a buried mode that allows octane to maximise the hydrophobic interactions with multiple residues in the active site (L56, L60, F63, F154 and L206). Within this conformation, C4/ C3 and C2 are accessible to Cpd I for the homolytic hydrogen abstraction from the C-H bond (Fig.…”

“…13,14 Also, recent work in our laboratory addressed this issue by creating chemodivergent variants of MthUPO from Myceliophthora thermophila for benzylic or aromatic oxyfunctionalisations. 30 To harness the impressive activities of UPOs for chemo-or regioselective transformations, protein engineering approaches and directed evolution techniques are of particular interest. Directed evolution consists of random or semi-rational mutagenesis to create enzyme libraries assessed for improved enzyme abilities by high throughput analysis.…”

Improving the Heterologous Production of Fungal Peroxygenases through an Episomal Pichia pastoris Promoter and Signal Peptide Shuffling System

“…UPOs are solely relying on hydrogen peroxide as cosubstrate for the formation of the reactive oxyferryl (Compound I) intermediate. In contrast to the well-known cytochrome P450 enzymes, they do not rely on auxiliary electron transport proteins and expensive cofactors. , Recent studies demonstrate impressive activities on a wide variety of structurally diverse substrates. − …”