A Promiscuous Cytochrome P450 Hydroxylates Aliphatic and Aromatic C−H Bonds of Aromatic 2,5‐Diketopiperazines

Abstract

Cytochrome P450 enzymes generally functionalize inert C−H bonds, and thus, they are important biocatalysts for chemical synthesis. However, enzymes that catalyze both aliphatic and aromatic hydroxylation in the same biotransformation process have rarely been reported. A recent biochemical study demonstrated the P450 TxtC for the biosynthesis of herbicidal thaxtomins as the first example of this unique type of enzyme. Herein, the detailed characterization of substrate requirements and biocatalytic applications of TxtC are reported. The results reveal the importance of N‐methylation of the thaxtomin diketopiperazine (DKP) core on enzyme reactions and demonstrate the tolerance of the enzyme to modifications on the indole and phenyl moieties of its substrates. Furthermore, hydroxylated, methylated, aromatic DKPs are synthesized through a biocatalytic route comprising TxtC and the promiscuous N‐methyltransferase Amir_4628; thus providing a basis for the broad application of this unique P450.

Biocatalytic synthesis of peptidic natural products and related analogues

“…Coupled with TB14, the three-enzyme reaction synthesized 36 thaxtomin D analogues ( Jiang et al., 2018 ). Finally, we generated a self-sufficient TxtC variant TCB14 and used it to produce 58 hydroxylated thaxtomin analogues and 43 hydroxylated desnitro thaxtomin analogues ( Jiang et al., 2018 , 2019 ). The use of multiple native and engineered NRPS modules for the synthesis of NRP analogues from simple building blocks was further demonstrated in vitro in the synthesis of antibiotics tyrocidine A and 10 analogues ( Niquille et al., 2021 ) ( Figure 4 B).…”

Enzymkatalysierte späte Modifizierungen: Besser spät als nie

“…Das Wildtypenzym P450 TxtC ermçglicht aliphatische sowie aromatische C-H-Hydroxylierungeni nd er späten Funktionalisierung eines Diketopiperazins (18). [57,58] Zudem berichten aktuelle Beispiele über die Dihydroxylierung zweier aliphatischer oder aromatischer C-H-Bindungen innerhalb eines Vitamin-D2-Motivs bzw.A rens: [59,60] Wildtyp-CYP109E1 hydroxyliert Vitamin D2 hochgradig regio-und stereoselektiv in einer Zwei-Schritt-Dihydroxylierung (Abbildung 7). [59] Darüber hinaus wurden P450-Enzyme im Hinblick auf multiple Oxyfunktionalisierungen konstruiert.…”

Interactive regulation between aliphatic hydroxylation and aromatic hydroxylation of thaxtomin D in TxtC: a theoretical investigation

“…1,2 Thxs are often pursued as herbicides for crop protection due to their virulence. 3,4 However, the synthetic routes to Thxs suffered from the lack of stereo-control and low overall yield. 5,6 Recently, it was reported that TxtC is able to effectively produce thaxtomin A, an herbicide approved by the U.S Environmental Protection Agency (EPA), 3,4,7 from thaxtomin D. TxtC is a member from cytochrome P450 family which consists of versatile monooxygenases to execute a broad array of biochemical transformations, such as, hydroxylation of alkane, oxidation of heteroatoms or aromatics, and dealkylation reactions.…”

“…3,4 However, the synthetic routes to Thxs suffered from the lack of stereo-control and low overall yield. 5,6 Recently, it was reported that TxtC is able to effectively produce thaxtomin A, an herbicide approved by the U.S Environmental Protection Agency (EPA), 3,4,7 from thaxtomin D. TxtC is a member from cytochrome P450 family which consists of versatile monooxygenases to execute a broad array of biochemical transformations, such as, hydroxylation of alkane, oxidation of heteroatoms or aromatics, and dealkylation reactions. [8][9][10][11][12] In these catalytic reactions, the active high-valent iron-oxo porphyrin πcation radical (Cpd I, as shown in Scheme 1A) species serves as the oxygen source.…”

“…The conversion from thaxtomin D to thaxtomin A in TxtC is a two-step process. Firstly, Cpd I inserts an oxygen atom into the aliphatic C14-H bond of thaxtomin D to generate thaxtomin B, which is followed by the second hydroxylation step on the meta-position of phenyl group of thaxtomin B to produce thaxtomin A 3,4,7 (see in Scheme 1B). It is worth noting that aliphatic and aromatic C-H bond usually have as high as 10～20 kcal/mol energy difference in the bond dissociation energies (BDE), which means that TxtC is able to use distinct strategies to accomplish the sequential aliphatic and aromatic hydroxylation on thaxtomin D. The catalytic hydroxylation mechanism of cytochrome P450 has been extensively investigated by both experimental and theoretical studies.…”