Developing biocatalysts for the production of nitroaromatics

Aromatic nitration is an immensely important industrial process to produce chemicals for a variety of applications. However, this acid-based electrophilic reaction is earth-damaging and often generates multiple unwanted impurities. Enzymes as biocatalysts are of great synthetic significance due to their typically high stereo-, regio- and chemo-selectivity. Recently, a unique cytochrome P450 enzyme, TxtE, was discovered to nitrate the indole of l-tryptophan within the biosynthetic pathway of phytotoxin thaxtomin. The aim of this work is to develop TxtE as practical direct nitration biocatalysts, thereby overcoming the major limitations of chemical nitration. Specifically, we created three artificial self-sufficient biocatalysts by fusing TxtE with the reductase domains of CYP102A1 (P450BM3) from Bacillus megaterium and of P450RhF from Rhodococcus sp. Recombinant fusion proteins were purified and biochemically characterized in terms of folding, coupling, total turnover numbers, thermostability, and optimal reaction conditions. The most active fusion enzyme, TxtE-BM3R, was then employed to nitrate unnatural substrates 4-F-dl-tryptophan and 5-F-l-tryptophan. Structural analyses of isolated products revealed enzyme’s altered regio-selectivity. Finally, we engineered TxtE’s active site by alanine scanning and identified multiple residues that controlled the activity and substrate specificity of biocatalysts. 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 including pharmacueticals, pesticides, explosives, and dyes.