Engineered P450 biocatalysts show improved activity and regio-promiscuity in aromatic nitration

Abstract

Nitroaromatics are among the most important and commonly used chemicals but their production often suffers from multiple unsolved challenges. We have previously described the development of biocatalytic nitration processes driven by an engineered P450 TxtE fusion construct. Herein we report the creation of improved nitration biocatalysts through constructing and characterizing fusion proteins of TxtE with the reductase domain of CYP102A1 (P450BM3, BM3R). The majority of constructs contained variable linker length while one was rationally designed for optimizing protein-protein interactions. Detailed biochemical characterization identified multiple active chimeras that showed improved nitration activity, increased coupling efficiency and higher total turnover numbers compared with TxtE. Substrate promiscuity of the most active chimera was further assessed with a substrate library. Finally, a biocatalytic nitration process was developed to nitrate 4-Me-DL-Trp. The production of both 4-Me-5-NO₂-L-Trp and 4-Me-7-NO₂-L-Trp uncovered remarkable regio-promiscuity of nitration biocatalysts.

Figure 1

Schematic depiction of chimeric TxtE-BM3R constructs with variable linker length or a swapped loop (yellow). The structure of human NADPH–cytochrome P450 reductase (PDB: 3QE2, right) represented not-available BM3R structure along with TxtE (PDB: 4TPO, left). The 25-AA linker of P450BM3 is shown as a green dash line (middle) along with the amino acid sequence.

Figure 2

Relative nitration activity of TxtE and chimeric TxtE-BM3R variants. All reactions contained 0.5 mM Trp and 1.5 µM P450. The TxtE reaction was further supplemented with 0.43 µM spinach Fer and 0.33 µM Frd. The reactions were incubated at 20 °C, 300 rpm for 30 minutes. All experiments were repeated at least three times. The results of TB12, TB13, TB15, and TB16 were shown as black bars. Chimeras showed significant difference in nitration activity compared to the wild type TxtE were indicated as *P < 0.05 or **P < 0.01.

Figure 3

TxtE and TB14 nitrated Trp and its analogues to varying degrees. The reactions were prepared as described previously and incubated at 20 °C, 300 rpm for 45 minutes. All experiments were repeated at least three times. Substrates showed significant difference in the TxtE and TB14 reactions were indicated with *P < 0.05 or **P < 0.01.

Figure 4

Both 4-Me-5-NO₂-l-Trp and 4-Me-7-NO₂-l-Trp were produced in the TB14 reaction with 4-Me-dl-Trp as substrate. The aromatic region of ¹H NMR spectrum of isolated product showed the chemical shifts of two sets of aromatic protons. Integrated values of these protons were also included.