Product Distributions of Cytochrome P450 OleTJE with Phenyl-Substituted Fatty Acids: A Computational Study

Abstract

There are two types of cytochrome P450 enzymes in nature, namely, the monooxygenases and the peroxygenases. Both enzyme classes participate in substrate biodegradation or biosynthesis reactions in nature, but the P450 monooxygenases use dioxygen, while the peroxygenases take H2O2 in their catalytic cycle instead. By contrast to the P450 monooxygenases, the P450 peroxygenases do not require an external redox partner to deliver electrons during the catalytic cycle, and also no external proton source is needed. Therefore, they are fully self-sufficient, which affords them opportunities in biotechnological applications. One specific P450 peroxygenase, namely, P450 OleTJE, reacts with long-chain linear fatty acids through oxidative decarboxylation to form hydrocarbons and, as such, has been implicated as a suitable source for the biosynthesis of biofuels. Unfortunately, the reactions were shown to produce a considerable amount of side products originating from Ca and Cb hydroxylation and desaturation. These product distributions were found to be strongly dependent on whether the substrate had substituents on the Ca and/or Cb atoms. To understand the bifurcation pathways of substrate activation by P450 OleTJE leading to decarboxylation, Ca hydroxylation, Cb hydroxylation and Ca-Cb desaturation, we performed a computational study using 3-phenylpropionate and 2-phenylbutyrate as substrates. We set up large cluster models containing the heme, the substrate and the key features of the substrate binding pocket and calculated (using density functional theory) the pathways leading to the four possible products. This work predicts that the two substrates will react with different reaction rates due to accessibility differences of the substrates to the active oxidant, and, as a consequence, these two substrates will also generate different products. This work explains how the substrate binding pocket of P450 OleTJE guides a reaction to a chemoselectivity.

Design of a H2O2‐generating P450SPα fusion protein for high yield fatty acid conversion

“…Electrons for these reactions are usually provided by NAD(P)H and transferred to the heme cofactor via different redox partners 15–18 . Although many P450 are redox partner‐dependent monoxygenase, it was reported that a recently identified subfamily of P450s, named as CYP152, is able to use H 2 O 2 as unique source of oxygen and electrons to catalyze the α or β hydroxylation or the oxidative decarboxylation of fatty acids, hence acting as peroxygenases 19–22 . Indeed, the generally accepted catalytic mechanism of this subfamily of enzymes requires the carboxylic group of the substrate to activate the H 2 O 2 and generate the ferryl‐oxo cation radical (Compound I), which is responsible for the fatty acid carbon α or β hydrogen abstraction and consequent hydroxylation or decarboxylation 23,24 .…”

“…[15][16][17][18] Although many P450 are redox partnerdependent monoxygenase, it was reported that a recently identified subfamily of P450s, named as CYP152, is able to use H 2 O 2 as unique source of oxygen and electrons to catalyze the α or β hydroxylation or the oxidative decarboxylation of fatty acids, hence acting as peroxygenases. [19][20][21][22] Indeed, the generally accepted catalytic mechanism of this subfamily of enzymes requires the carboxylic group of the substrate to activate the H 2 O 2 and generate the ferryl-oxo cation radical (Compound I), which is responsible for the fatty acid carbon α or β hydrogen abstraction and consequent hydroxylation or decarboxylation. 23,24 Sphingomonas paucimobilis CYP152B1 (P450 SPα ) catalysis is described to exclusively yield the α hydroxylation of fatty acid, exploiting only hydrogen peroxide and not requiring expensive external co-substrate addition or engagement of electron shuttles, [25][26][27] therefore it is an interesting candidate for applicative exploitation as biocatalyst.…”

Unraveling the Catalytic Mechanism of Taxadiene-5α-hydroxylase from Crystallography and Computational Analyses

“…The QM/MM coupling was treated by the electronic embedding scheme, while the QM/MM boundary was treated with hydrogen link atoms with the charge-shift model. UB3LYP functionals were used for the QM region, − which were demonstrated to be reliable for Fe-containing enzymes. ,− The double-ζ basis set def2-SVP­(B1) was used to optimize the geometry, while the larger basis set of def2-TZVP­(B2) was used to correct the energies . Grimme’s D3 method was employed to account for dispersion corrections in all quantum mechanical calculations. − The QM region in the system included the substrate, the propionate-truncated heme, and the side chain of Cys383.…”

Decarboxylation in Natural Products Biosynthesis

“…Initially discovered in the genome of the bacteria Jeotgalicoccus spp., OleT JE has been found to generate terminal olefins such as 18-methyl-1-nonadecene and 17-methyl-1-nonadecene . Noncanonical substrates, such as short-chain aliphatic or phenyl-substituted fatty acids, yield Cα and Cβ hydroxyl products. − The regio- and chemoselectivity of the reaction depend on multiple factors, including the structure of the substrate, the positioning of the substrate within the active site, and the accessibility of the oxidizing species Fe IV =O to the substrate.…”