To continue with the mutagenesis approach, we grouped two amino acid residues and saturated them simultaneously with a reduced codon degeneracy (NDT), thereby obtaining double mutants with improved TONs (Tab. S4). The improved double mutated variants were L60F/F154I and L60F/F154V (1.2-fold improvement compared to the wildtype, Tab. S4). The residues from the single and double saturation libraries, which had a positive or neutral influence on the NBD conversion (Tab. S4) were selected for recombination. These residues were F59Q, L60F/Q/M, A57I, F154I/V, E158D, S159N/G and A161I/F. Inspired by Reetz' single-, double-, and triple-code saturation mutagenesis approaches, we chose to recombine all residues and variations in all combinatorial possibilities including the respective wildtype amino acid residue. This combination led to 864 mutants and required the screening of more than 2,300 transformants. The recombination library resulted in the discovery of triple and quadruple mutations with up to 16.5-fold improved catalytic efficiency (kcat/Km, Tab. 2). All of the most active variants harboured amino acid exchanges at the L60 position (L60F/M) and the mutation S159G. The kinetic measurements revealed 8.2-fold (F59Q/L60F/S159G), 10.8fold (F59Q/L60M/S159G/F154A) and 16.5-fold (L60F/S159G/A161F) increased kcat/Km values relative to the wildtype (Tab. 2). Whereas the Km value of NBD was decreased or similar to the wildtype, the values were significantly decreased for H2O2 (Tab. S5). The kcat for NBD, however, was substantially improved for the identified triple and quadruple mutants with an 18.6-fold increase for the variant L60F/S159G/A161F relative to the wildtype. Docking calculations were used to obtain starting points for MD simulations in order to analyse the binding of NBD in the wildtype and the variants L60F and L60F/S159G/A161F (Figs. 1, S13 and S14). These simulations revealed a switch in the binding mode of NBD when moving from wildtype to L60F and L60F/S159G/A161F (Fig. ). Due to the inclusion of bulkier residues in the inner active site (first L60F, then A161F), the NBD substrate was reoriented from its original more buried binding pose in the wildtype to partially occupying the entrance channel in L60F/S159G/A161F. This new binding pose allows the NBD to better approach the Fe=O active species in a near attack conformation, thus facilitating the oxidation reaction (Fig. ). To determine the influence of the engineered variants on the regio-and chemoselectivity, we investigated the oxyfunctionalisation of naphthalene and its derivatives (Scheme 1). The most active variants were selected, and an initial screening was performed by GC-MS, resulting in significant changes in the selectivity (Fig. ). Utilising a syringe pump setup achieved more than 10,000 TONs with variant F59Q/L60F/S159G for the conversion of naphthalene to 1,4-naphthoquinone 1a (Scheme 2a). These TONs represent a 10-fold increase to the previously published MthUPO wildtype activity. By increasing H2O2 equivalents, 2-hydroxy-1,4-naphthoquinone 1b was formed as a byproduct, which is a natural dye known as Lawsone. The biotransformation of 2-methylnaphthalene with the wildtype led predominantly to 6-methyl-1,4-naphthoquinone (Fig. ). Variant L60F/S159G/A161F was able to shift the major product formation to 2-methyl-1,4-naphthoquinone Scheme 1: Catalytic activity of MthUPO variants for the hydroxylation of naphthalene and its derivatives (reaction conditions are shown in Tab. S6, standard deviation <5.2 %) (2a), also known as vitamin K3, demonstrating the regioselectivity of this variant (Scheme 2b). We were pleased to note that we could also identify a variant with the preference for the methyl hydroxylation of 2methylnaphthalene. Variant L60F showed an altered chemoselectivity dramatically suppressing the aromatic hydroxylation and accessing the hydroxylation of the methyl group (2b) as the major product. Also the overoxidation to the aldehyde was observed (2c, Scheme 2b). MD simulations with the variant L60F and 2-methylnaphthalene demonstrated that only the methyl group was able to approach the active Fe=O species in a catalytically competent pose due to the presence of the bulky phenylalanine residue L60F (Fig. ). When 2methylnaphthalene was bound into the variant L60F/S159G/A161F (Fig. ), however, a binding pose similar to the previously observed for NBD in this variant was observed (Figs. and). This binding mode is promoted by hydrophobic interactions occurring in the newly engineered active site, which is dominated by the presence of several aromatic residues (F59, F60, F63, F154, and F161). Within this new binding pose, the substituted aromatic ring 1 of 2-methylnaphthalene is placed close enough to the Fe=O catalytic species to react while keeping the 2-methyl group away from it. This different binding mode of the L60F variant is responsible for the observed switch in chemoselectivity (Figs. and). When the methyl substitution was changed from 2-methyl to 1-methylnaphthalene a decrease in the activity of 50 % was observed yielding to the opposite unsubstituted ring 2 oxidation (Scheme 2c). More surprisingly, also substitutions in position 2 at the naphthalene led to the oxidation of the unsubstituted ring generating the products 6-methoxy-(4a, Scheme 2d) and 6-bromo-1,4-naphthoquinone (5a, Scheme. 2e) with diminished TONs relative to 2-methylnaphthalene. For 2-bromo-naphthalene also 2-bromo-1,4-naphthoquinone (5b) was detected as a by-product. MD simulations with variant L60F/S159G/A161F and 2-methoxy-naphthalene (Fig. ) revealed a preferential binding in a catalytically competent pose equivalent to the binding mode observed for 2-methyl naphthalene (Fig. ). However, the bulky methoxy group at the 2-position sterically clashes with the L60F residue, and the naphthalene core needs to rotate slightly when approaching the Fe=O active species to place the 2-methoxy group away from the L60F and the heme cofactor. This rotation brings the unsubstituted aromatic ring 2 closer to the Fe=O active species, as opposite to 2methylnaphthalene (Fig. ), inducing a switch in the regioselectivity (Fig. ). A similar behaviour was also observed for 1-methylnaphthalene when bound in variant L60F/S159G/A161F in a catalytically competent pose (Fig. ). Intrigued by the chemoselective benzylic hydroxylation of L60F, we tested this variant with indane and tetralin (1,2,3,4tetrahydronaphthalene). L60F was able to convert indane with more than 8,000 TONs and an improved enantioselectivity for the (R)-1-indanol (5a) from 85 % ee (wildtype) to 95 % ee (Tab. 3 and S7). Interestingly, further examination of the most active variants L60F/S159G/A161F and F59Q/L60M/S159G/F154A revealed the excess in formation of the (S)-enantiomer (Tab. S7). For the bioconversion of tetralin to the alcohol 6a, an improved enantioselectivity for the (R)-enantiomer was detected for L60F (74 % ee) relative to the wildtype (45 % ee). Similar to indane, a variant could be identified, which forms predominantly the (S)enantiomer (F59Q/L60M/S159G/F154A). The overoxidation to the ketone as a major product was achieved by the same variant with TONs of 440. MD simulations with indane bound within the L60F variant (Fig. ) characterised a preferential binding pose of the substrate that resembles the previously observed pose for NDB and 2-methylnaphthalene in L60F/S159G/A161F (Figs. and). In this case, indane mainly interacts with the aromatic rings of F63 and L60F, establishing C-H•••π interactions. These hydrophobic interactions keep the substrate in a preferred binding pose where only the pro-R 1-H-indane is close enough and well-aligned to the Fe=O active species to be efficiently hydroxylated (Fig. ). In the present work, the novel MthUPO was engineered using S. cerevisiae as expression host and NBD as an assay system. By initial saturation of relevant amino acid residues in single and double saturation mutageneses, key residues were identified. This mutagenesis also led to an increased understanding of the highly conserved catalytic glutamate, its stabilisation by histidine, and-most surprisingly-the generation of the active variant E158D.