Computational-Aided Engineering of a Selective Unspecific Peroxygenase toward Enantiodivergent β-Ionone Hydroxylation

Abstract

Unspecific peroxygenases (UPOs) perform oxyfunctionalizations for a wide range of substrates utilizing H2O2 without the need for further reductive equivalents or electron transfer chains. Tailoring these promising enzymes toward industrial application was intensely pursued in the last decade with engineering campaigns addressing the heterologous expression, activity, stability, and improvements in chemo- and regioselectivity. One hitherto missing integral part was the targeted engineering of enantioselectivity for specific substrates with poor starting enantioselectivity. In this work, we present the engineering of the short-type MthUPO toward the enantiodivergent hydroxylation of the terpene model substrate, β-ionone. Guided by computational modeling, we designed a small smart library and screened it with a GC–MS setup. After two rounds of iterative protein evolution, the activity increased up to 17-fold and reached a regioselectivity of up to 99.6% for the 4-hydroxy-β-ionone. Enantiodivergent variants were identified with enantiomeric ratios of 96.6:3.4 (R) and 0.3:99.7 (S), respectively.

Peroxygenase-Catalyzed Allylic Oxidation Unlocks Telescoped Synthesis of (1S,3R)-3-Hydroxycyclohexanecarbonitrile

“…Intermediate 3 not being commercially available, a C–H oxyfunctionalization step is required to introduce the ketone functional group into the commercially available pro-chiral starting material 1 . Regioselective allylic oxidation of alkenes, in particular substituted cyclohexenes, remains challenging and reported chemical strategies include the use of hypervalent iodide reagents, transition metal catalysis, , or photocatalysis. , Enzymatic strategies are underdeveloped; examples on few selected substrates include the use of cytochrome P450s and unspecific peroxygenases (UPOs). , UPOs are well known to selectively catalyze the oxyfunctionalization of ethylbenzene at the benzylic position to the corresponding enantiopure ( R )-1-phenylethanol, and can also further oxidize both enantiomers of 1-phenylethanol to acetophenone, yet remain to be further explored with substituted cyclohexene substrates, in particular with regard to stereoselectivity. , …”

Understanding the Multifaceted Mechanism of Compound I Formation in Unspecific Peroxygenases through Multiscale Simulations

“…2,9 For many substrates (such as n-alkanes), UPOs typically exhibit high selectivity in the activation of C−H bonds. 10,11 For example, a previous study by Peter et al 10 reported a comprehensive examination of the catalytic selectivity of UPO found in the fungus Agrocybe aegerita (AaeUPO, Figure 1) 12 for the hydroxylation of alkanes. 10 Several substrates were explored, including propane, n-butane, n-pentane, n-hexene, nheptane, and n-octane.…”

“…The unspecific peroxygenases (UPOs) are an emerging class of enzymes capable of selective oxyfunctionalization of unactivated hydrocarbons in nonaqueous environments under mild conditions, − making them promising biocatalysts for efficient transformation of chemical products in feedstocks. The term “unspecific” refers to the promiscuous nature of UPOs and their broad spectrum of substrates. , These substrates encompass linear, branched, and cyclic alkanes/alkenes, as well as aromatic and heterocyclic compounds and others. , For many substrates (such as n -alkanes), UPOs typically exhibit high selectivity in the activation of C–H bonds. , For example, a previous study by Peter et al reported a comprehensive examination of the catalytic selectivity of UPO found in the fungus Agrocybe aegerita ( Aae UPO, Figure ) for the hydroxylation of alkanes . Several substrates were explored, including propane, n -butane, n -pentane, n -hexene, n -heptane, and n -octane.…”

Computational Characterization of the Reactivity of Compound I in Unspecific Peroxygenases

“…Given these advantages, UPOs stand out as promising next-generation biocatalysts for the sustainable conversion of feedstocks into valuable products. As such, since the initial discovery of UPO in the fungus Agrocybe aegerita ( Aae UPO), a wide range of UPO variants have been identified and engineered. − ,− …”

“…For instance, the hydroxylation of n -alkanes by Aae UPO yields solely 2- and 3-alcohols It remains elusive how the enzyme–substrate interactions, the chemical reactivity of various reaction sites, and the protein’s electrostatic environment culminate in the regio- and enantioselectivity of hydroxylation. Although recent studies have employed molecular simulations to understand the catalytic selectivity , and facilitate the directed evolution of UPOs, − these simulations fell short of systematic quantum mechanical (QM) characterizations of substrate reactivities in realistic enzyme environments. Notably, no previous studies characterized the free energy profiles of UPO-catalyzed reactions at QM level of theory, which are essential for understanding the chemical reactivity in condensed-phase biological systems. − As established in our prior study, relying solely on potential energy calculations can be misleading due to their sensitivity to the initial conditions (ICs) in intricate biological systems.…”