An artificial self‐sufficient cytochrome P450 directly nitrates fluorinated tryptophan analogs with a different regio‐selectivity

Abstract

Aromatic nitration is an immensely important industrial process to produce chemicals for a variety of applications, but it often suffers from multiple unsolved challenges. Enzymes as biocatalysts have been increasingly used for organic chemistry synthesis due to their high selectivity and environmental friendliness, but nitration has benefited minimally from the development of biocatalysis. In this work, we aimed to develop TxtE as practical biocatalysts for aromatic nitration. TxtE is a unique class I cytochrome P450 enzyme that nitrates the indole of l-tryptophan. To develop cost-efficient nitration processes, we fused TxtE with the reductase domains of CYP102A1 (P450BM3) and of P450RhF to create class III self-sufficient biocatalysts. The best engineered fusion protein was comparable with wild type TxtE in terms of nitration performance and other key biochemical properties. To demonstrate the application potential of the fusion enzyme, we nitrated 4-F-dl-tryptophan and 5-F-l-tryptophan in large scale enzymatic reactions. Tandem MS/MS and NMR analyses of isolated products revealed altered nitration sites. To our knowledge, these studies represent the first practice in developing biological nitration approaches and lay a solid basis to the use of TxtE-based biocatalysts for the production of valuable nitroaromatics.

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

“…We previously reported three self-sufficient TxtE constructs 22 . Of the two reductase domains that we evaluated, the di-flavin reductase BM3R homologous to eukaryotic cytochrome P450 reductase 32 conferred superior TxtE nitration activity compared with the P450RhF reductase domain (RhFRED), a natural fusion of Frd and Fer 33 .…”

“…In these applications, enzymes are capable of catalysing a breadth of organic transformations 20 . However, despite Nature’s successful application of several strategies to synthesize nitro-containing compounds 21 , there has been comparably little effort to exploit them in chemical industry 22 .…”

“…However, the requirement of auxiliary redox proteins and low activity and electron coupling efficiency are common limitations that constrain frequent industrial implementation of P450s 25, 26 . Inspired by previous research on artificial self-sufficient systems 27–30 , we have recently addressed the need of redox partners in TxtE applications 22 by fusing it with the reductase domain (BM3R) of naturally self-sufficient P450 P450BM3 31 . The chimera outperformed TxtE supplemented with spinach ferredoxin (Fer) and ferredoxin reductase (Frd) in terms of catalytic activity, and was subsequently utilized in the biocatalytic syntheses of two fluorinated nitro-Trp analogues 22 .…”

“…Inspired by previous research on artificial self-sufficient systems 27–30 , we have recently addressed the need of redox partners in TxtE applications 22 by fusing it with the reductase domain (BM3R) of naturally self-sufficient P450 P450BM3 31 . The chimera outperformed TxtE supplemented with spinach ferredoxin (Fer) and ferredoxin reductase (Frd) in terms of catalytic activity, and was subsequently utilized in the biocatalytic syntheses of two fluorinated nitro-Trp analogues 22 . However, both electron coupling efficiency and total turnover number (TTN) of the developed chimeric enzyme were 20% lower than wild type TxtE 22 .…”

“…The chimera outperformed TxtE supplemented with spinach ferredoxin (Fer) and ferredoxin reductase (Frd) in terms of catalytic activity, and was subsequently utilized in the biocatalytic syntheses of two fluorinated nitro-Trp analogues 22 . However, both electron coupling efficiency and total turnover number (TTN) of the developed chimeric enzyme were 20% lower than wild type TxtE 22 . These must be addressed to attain our goal of developing efficient TxtE-based nitration biocatalysts for biotechnological applications.…”