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 S. cerevisiae with the novel MthUPO. 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 cat /K m 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 % ee. Additionally, the first reported effective exchange of the conserved catalytic Glu residue was observed. File list (2) download file view on ChemRxiv Manuscript_Knorrscheidt et al.pdf (566.94 KiB) download file view on ChemRxiv Supporting_Knorrscheidt et al.pdf (5.26 MiB)

Peroxide-Mediated Oxygenation of Organic Compounds by Fungal Peroxygenases

“…A modified Saccharomyces - Pichia system has recently been applied to express four active peroxygenases (r Mro UPO, r Cgl UPO, r Mth UPO, and r Tte UPO; Table 1 ) using a ‘Golden Gate platform’ consisting of three modules (signal peptide library—module 1, UPO genes—module 2, and protein tags—module 3) [ 65 , 66 ]. The same group used the Saccharomyces system to engineer one of the newly expressed enzymes (r Mth UPO) from the sordariomycete Myceliophthora thermophila ( Corynascus heterothallicus ) [ 67 ].…”

Oxygenating Biocatalysts for Hydroxyl Functionalisation in Drug Discovery and Development

“…Additionally, a fter transferring expression to Pichia pastoris , Mth UPO was used on a preparative scale to catalyse the hydroxylation of N‐ phthaloyl‐phenethylamine to produce 2‐ N‐ phthaloyl‐1‐phenylethanol in 57 % yield, 99 % ee [152] . The group have extended this methodology for the engineering of Mth UPO in S. cerevisiae [153,154] . Consequently, Mth UPO variant A161L was found to catalyse octane hydroxylation, with selectivity towards terminal 1‐octanol (38 %).…”

Redesigning Enzymes for Biocatalysis: Exploiting Structural Understanding for Improved Selectivity

“…For mutant L60F, introducing a bulky phenylalanine allowed only the methyl group to get close enough to the catalytic center to be oxidized. Additional mutations, L60F/S159G/A161F, increased hydrophobicity, pushing the naphthyl aromatic ring moiety toward the heme-iron center, to afford a different quinone product (Knorrscheidt et al, 2021) (Figure 8C).…”