Exploring the Role of Phenylalanine Residues in Modulating the Flexibility and Topography of the Active Site in the Peroxygenase Variant PaDa-I

Abstract

Unspecific peroxygenases (UPOs) are fungal heme-thiolate enzymes able to catalyze a wide range of oxidation reactions, such as peroxidase-like, catalase-like, haloperoxidase-like, and, most interestingly, cytochrome P450-like. One of the most outstanding properties of these enzymes is the ability to catalyze the oxidation a wide range of organic substrates (both aromatic and aliphatic) through cytochrome P450-like reactions (the so-called peroxygenase activity), which involves the insertion of an oxygen atom from hydrogen peroxide. To catalyze this reaction, the substrate must access a channel connecting the bulk solution to the heme group. The composition, shape, and flexibility of this channel surely modulate the catalytic ability of the enzymes in this family. In order to gain an understanding of the role of the residues comprising the channel, mutants derived from PaDa-I, a laboratory-evolved UPO variant from Agrocybe aegerita, were obtained. The two phenylalanine residues at the surface of the channel, which regulate the traffic towards the heme active site, were mutated by less bulky residues (alanine and leucine). The mutants were experimentally characterized, and computational studies (i.e., molecular dynamics (MD)) were performed. The results suggest that these residues are necessary to reduce the flexibility of the region and maintain the topography of the channel.

Identification and Expression of New Unspecific Peroxygenases – Recent Advances, Challenges and Opportunities

“…HPLC-and GC-dependent systems generally require long analysis time. The development of efficient screening methods to identify new UPOs of currently uncharacterized strains and to screen mutant libraries is thus -Espeja et al, 2015-Espeja et al, , 2019-Espeja et al, , 2016aGómez de Santos et al, 2018;Martin-Diaz et al, 2018;Ramirez-Escudero et al, 2018;Ramirez-Ramirez et al, 2020 needed. An outstanding approach includes a directed-evolution step coupled to a multiple injections in a single experimental run (MISER)-GC-MS-system (Knorrscheidt et al, 2019(Knorrscheidt et al, , 2020.…”

“…More than 400 substrates are now known to be converted by UPOs ( Hobisch et al, 2020 ). These reactions range from aromatic oxidations ( Ramirez-Ramirez et al, 2020 ), epoxidation of aromatics ( Zhang et al, 2021 ), oxidation of alcohols ( Kiebist et al, 2017 ), dealkylation ( Püllmann and Weissenborn, 2021 ), to oxygenation of unsaturated fatty acids ( Linde et al, 2020 ) and halogenations ( Ullrich et al, 2004 ).…”

“…In addition, two wild-type UPOs from the ascomycetes Collariella virescens (syn., Chaetomium virescens ) and Daldinia caldariorum were successfully expressed in E. coli (2.8–7 mg L –1 in culture), purified, and catalytically characterized ( Linde et al, 2020 ). Furthermore, for characterization and modulation of the topography of the active site in UPO enzymes, site-directed mutagenesis and subsequent expression in S. cerevisiae revealed the importance of heme channel residues, such as amino acid positions 76, 191 and 241, for substrate accessibility and catalytic activity ( Gómez de Santos et al, 2019 ; Ramirez-Ramirez et al, 2020 ). In particular, substitutions F191S and G241D in an evolved r Aae UPO variant enhanced hydroxylation efficiency toward propranolol ( Gómez de Santos et al, 2018 ), while replacement of F76 by a small residue like alanine had a negative effect on substrate affinity ( Ramirez-Ramirez et al, 2020 ).…”

Ether Oxidation by an Evolved Fungal Heme-Peroxygenase: Insights into Substrate Recognition and Reactivity

“…Acetonitrile for HPLC analysis and salts for buffer solutions were obtained from JT Baker. PaDa-I was heterologously expressed and purified [14] to >95% of electrophoretic homogeneity and a Reinheitszahl value (R z = A 407 nm/A 280 nm) of 2.1. Culture media reagents were purchased from Difco.…”

“…The PaDa-I crystallographic structure model (PDB ID: 5OXU) was used to run a 50 ns molecular dynamics simulation as previously described [14] (Ramirez-Ramirez et al, 2020). A total of 243 frames were extracted from the last 20 ns of the simulation, to obtain different conformations of the enzyme for the molecular docking with the substrates.…”