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

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.

), H 2 O 2 concentration was varied as depicted in Figure  S3. Measurement conditions: absorbance was measured at 425 nm for one hour in triplicates, values were calculated with the corrected extinction coefficient of 10870 M -1 *cm -1 .

Figure S7
GC-MS chromatogram (in selected ion monitoring mode with the depicted m/z traces) of the later described MthUPO variant of naphthalene and naphthalene derivatives, A) F59Q/L60F/S159G with naphthalene to 1,4naphthoquinone, B) F59Q/L60F/S159G with naphthalene to Lawsone product, C) L60F/S159G/A161F with 2methylnaphthalene to vitamin K 3 , D) L60F with 2-methylnaphthalene to 2-naphthalenemethanol and β-naphthaldehyde, E) L60F/S159G/A161F with 1-methylnaphthalene to 5-methyl-1,4-naphthoquinone, F) L60F/S159G/A161F with 2methoxynaphthalene to 6-methoxy-1,4-naphthoquinone and G) L60F/S159G/A161F with 2-bromonaphthalene to 6-bromo-1,4-naphthoquinone and 2-bromo-1,4-naphthoquinone.  Figure S9. Evolution of NBD catalytically relevant binding modes in wildtype, L60F and L60F/S159G/A161F variants as observed from MD simulations (see Figure S10). Mutated positions are highlighted in orange. NBD explores substantially different near attack conformations (NACs) in each variant due to the new introduced mutations. L60F displaces NBD from a more buried binding pose in wildtype, to a new binding mode that increases the aromatic interactions with residues F63 and newly introduced L60F. Finally, A161F and S159G mutations led to a significantly reduced active site that forces NBD to explore a new binding mode, perpendicular to the haem, that facilitates its interaction with the catalytic Fe=O species (see also Figure S10). Figure S10. Analysis of NBD binding modes through three independent MD replicas in: A) wildytpe; B) L60F variant; and C) L60F/S159G/A161F variant. Key distances relevant for hydroxylation and aromatic oxidation are monitored along MD simulations, as described in the schemes. Fe=O -H(CH) distance and angle of attack (O-H-C) are used as geometric parameters to characterise near attack conformations for effective C-H hydroxylation in heat maps. Representative snapshots from MD trajectories (highlighted with a "star" symbol) that describe reactive near attack conformations explored during MDs are shown. Distances and angles are given in angstroms (Å) and degrees (º), respectively. Angle vs. distance heat maps show that NBD explores more catalytically competent poses in L60F/S159G/A161F and L60F variants than in the WT, in line with the higher activity experimentally observed. The differences in the NBD binding poses in the three variants are discussed in Figure  S9.

Figure S11
Analysis of 2-methylnaphthalene binding modes through three independent MD replicas in L60F variant. Key distances relevant for hydroxylation and aromatic oxidations are monitored along MD simulations, as described in the schemes. Representative snapshots from MD trajectories (highlighted with a "star" symbol) that describe reactive near attack conformations explored during MDs are shown. Distances and angles are given in angstroms (Å) and degrees (º), respectively. 2-methylnaphthalene bound in L60F variant predominantly explores catalytically relevant binding poses in which the 2methyl group is placed in a near attack conformation respect to the Fe=O active species. Differences observed for naphthalene derivatives binding modes in different MthUPO variants are discussed in Figure S13.

Figure S12
Analysis of 2-methylnaphthalene binding modes through three independent MD replicas in L60F/S159G/A161F variant. Key distances relevant for hydroxylation and aromatic oxidations are monitored along MD simulations, as described in the schemes. Representative snapshots from MD trajectories (highlighted with a "star" symbol) that describe reactive near attack conformations explored during MDs are shown. Distances and angles are given in angstroms (Å) and degrees (º), respectively. 2-methylnaphthalene bound in L60F/S159G/A161F variant predominantly explores catalytically relevant binding poses in which the substituted aromatic ring is placed in a near attack conformation respect to the Fe=O active species. Differences observed for naphthalene derivatives binding modes in different MthUPO variants are discussed in Figure S13.

Figure S13
Differences in catalytically relevant binding modes of 2-substituted naphthalene derivatives (2-methlynaphthalene and 2-methoxynaphthalene) in L60F and L60F/S159G/A161F variants as observed from MD simulations (see Figure S11, S12 and S14). Mutated positions are highlighted in orange. 2-methlynaphthalene explores substantially different near attack conformations (NACs) in each variant due to the new introduced mutations. In L60F, 2-methlynaphthalene explores catalytically competent poses in which the 2-methyl group is suitable to directly interacts with Fe=O active species. On the other hand, in L60F/S159G/A161F variant, the substrate is displaced form the former binding position to a new one that resembles the binding mode observed by NBD in this triple mutant ( Figure S9). This new binding mode, induced by the presence of bulky A161F mutation, allows the direct interaction between the substituted aromatic ring of 2methlynaphthalene and the catalytic Fe=O species (see Figure S15). When 2-methoxynaphthalene is bound in L60F/S159G/A161F variant, it occupies the same binding position as 2-methyl derivative. However, because the more bulkier 2-methoxy group, the substrate slightly rotates and preferentially explores catalytically relevant conformations in which the 2-methoxy group is placed far from the haem group and the unsubstituted aromatic ring is placed closer to the Fe=O. Because of this reorientation, the regioselectivity of the oxidation reaction changes from preferential functionalisation at the substituted aromatic ring in 2-methylnaphtalene to the oxidation at the unsubstituted one in 2-methoxynaphthalene when L60F/S159G/A161F variant is used. Figure S14. Analysis of 2-methoxynaphthalene binding modes through three independent MD replicas in L60F/S159G/A161F variant. Key distances relevant for hydroxylation and aromatic oxidations are monitored along MD simulations, as described in the schemes. Representative snapshots from MD trajectories (highlighted with a "star" symbol) that describe reactive near attack conformations explored during MDs are shown. Distances and angles are given in angstroms (Å) and degrees (º), respectively.2-methoxynaphthalene bound in L60F/S159G/A161F variant predominantly explores catalytically relevant binding poses in which the unsubstituted aromatic ring is placed in a near attack conformation respect to the Fe=O active species. Differences observed for naphthalene derivatives binding modes in different MthUPO variants are discussed in Figure  S13. Figure S15. Analysis of 1-methylnaphthalene binding modes through three independent MD replicas in L60F/S159G/A161F variant. Key distances relevant for hydroxylation and aromatic oxidations are monitored along MD simulations, as described in the schemes. Representative snapshots from MD trajectories (highlighted with a "star" symbol) that describe reactive near attack conformations explored during MDs are shown. Distances and angles are given in angstroms (Å) and degrees (º), respectively. 1-methylnaphthalene bound in L60F/S159G/A161F variant predominantly explores catalytically relevant binding poses in which the unsubstituted aromatic ring is placed in a near attack conformation respect to the Fe=O active species.