Structural Insights into the Substrate Promiscuity of a Laboratory-Evolved Peroxygenase

Abstract

Because of their minimal requirements, substrate promiscuity and product selectivity, fungal peroxygenases are now considered to be the jewel in the crown of C−H oxyfunctionalization biocatalysts. In this work, the crystal structure of the first laboratory-evolved peroxygenase expressed by yeast was determined at a resolution of 1.5 Å. Notable differences were detected between the evolved and native peroxygenase from Agrocybe aegerita, including the presence of a full N-terminus and a broader heme access channel due to the mutations that accumulated through directed evolution. Further mutagenesis and soaking experiments with a palette of peroxygenative and peroxidative substrates suggested dynamic trafficking through the heme channel as the main driving force for the exceptional substrate promiscuity of peroxygenase. Accordingly, this study provides the first structural evidence at an atomic level regarding the mode of substrate binding for this versatile biocatalyst, which is discussed within a biological and chemical context.

Two New Unspecific Peroxygenases from Heterologous Expression of Fungal Genes in Escherichia coli

“…In conclusion, the structural-functional information on the two new UPOs supports the importance of active-site phenylalanines on UPO reactions with aromatic compounds (31,33). However, it clearly associates the higher activity on simple aromatics in these UPOs with the absence of a phenylalanine residue at the position equivalent to Phe121 in the A. aegerita UPO, while the opposite role has been proposed for this residue in the latter enzyme.…”

“…Among the host systems used for expressing UPO-encoding genes, production using A. oryzae is a proprietary technology of the company Novozymes (Bagsvaerd, Denmark), and therefore, the strains, vectors, and growth conditions are not publicly available. Yeast expression, as far as reported, requires previous directed evolution introducing mutations that result in structural differences with respect to the wild-type enzymes (33). Overexpression in E. coli as inclusion bodies followed by in vitro activation is routinely used for fungal (class II) peroxidases (34)(35)(36)(37)(38)(39), but a protocol for in vitro activation of UPOs from inclusion bodies has not been developed yet.…”

“…6A and B, respectively). Interestingly, a docking study of the A. aegerita UPO evolved for heterologous expression in yeast (the so-called PaDa-I variant) identified three of the above-mentioned phenylalanine residues (Phe121, Phe199, and, to a lesser extent, Phe69) as being involved in interaction with all the (aromatic) substrates assayed (33).…”

Benchmarking of laboratory evolved unspecific peroxygenases for the synthesis of human drug metabolites

“…The heme channel of AaeUPO is furnished with 9 aromatic residues, of which Phe76 and Phe191 define its access, while Phe69, Phe121 and Phe199 are involved in positioning the substrate for catalysis [17]. In the crystal structure of the PaDa-I variant recently made available, we noted that the F311L mutation is implicated in broadening the access channel, and it is also responsible for the dual conformational state of Phe191, conferring unheralded plasticity to the heme entrance in some of the evolved UPO variants [18]. Such flexibility at the heme access channel is not a feature of the native AaeUPO and accordingly, it is reasonable to think that this modifications might explain the improved yields with dextromethorphan (1) given its bulk.…”

Solvent‐Free Photobiocatalytic Hydroxylation of Cyclohexane

“…Cyclohexane was chosen as poorly water-soluble (0.06 gL À 1 , 0.7 mM at 20°C) model substrate, whereas the work dedicated to the evaluation of the photocatalyst was based on ethylbenzene (water solubility of 0.2 gL À 1 , 1.4 mM at 20°C), a standard substrate for UPO. PaDaÀ I is a laboratory-evolved variant of UPO from Agrocybe aegerita [11] with strong heterologous expression, high activity and stability. Hence, AaeUPO PaDaÀ I became the enzyme of choice for the here presented study.…”