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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.
Highlights
The nitro (-NO2) group acts as an essential unit in a number of pharmaceuticals[1], exemplified by anticancer drug nilutamine, antiparkinson agent tolcapone, and anti-infective agents chloramphenicol and the recently approved delamanid[2] and nifurtimox-eflornithine combination[3]
Inspired by previous research on artificial self-sufficient systems[27,28,29,30], 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 P450BM331
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 ferredoxin reductase (Frd) and Fer[33]
Summary
The nitro (-NO2) group acts as an essential unit in a number of pharmaceuticals[1], exemplified by anticancer drug nilutamine, antiparkinson agent tolcapone, and anti-infective agents chloramphenicol and the recently approved delamanid[2] and nifurtimox-eflornithine combination[3]. 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] Both electron coupling efficiency and total turnover number (TTN) of the developed chimeric enzyme were 20% lower than wild type TxtE22. These chimeras were developed by varying the length of a linker connecting TxtE (from Streptomyces scabies) and BM3R and swapping a putative interfacial loop on the TxtE to improve interactions with the reductase domain (Fig. 1) These studies have yielded TxtE-BM3R constructs with improved catalytic turnover, coupling efficiency, and broad substrate specificity. These advancements constitute essential steps on the path toward developing advanced nitration biocatalysts for industrial implementation
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