title: 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.
authors.affiliation: Department of Medicinal Chemistry, Center for Natural Products, Drug Discovery and Development, College of Pharmacy, University of Florida, Gainesville, Florida, 32610, USA.
authors.forename: Ran
authors.initials: R
authors.lastname: Zuo
authors.affiliation: Department of Medicinal Chemistry, Center for Natural Products, Drug Discovery and Development, College of Pharmacy, University of Florida, Gainesville, Florida, 32610, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Pharmaceutical Engineering, School of Chemical Engineering, Nanjing University of Science and Technology, Nanjing, Jiangsu, 210094, China.
authors.forename: Chao
authors.identifier: http://orcid.org/0000-0002-9228-1924
authors.initials: C
authors.lastname: Jiang
authors.affiliation: Department of Physiology and Biophysics and the Massey Cancer Center, Virginia Commonwealth University School of Medicine, Richmond, Virginia, 23298, USA.
authors.forename: John C
authors.initials: JC
authors.lastname: Hackett
authors.affiliation: Department of Plant Pathology, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida, 32611, USA.
authors.forename: Rosemary
authors.initials: R
authors.lastname: Loria
authors.affiliation: Department of Chemistry, University of Florida, Gainesville, Florida, 32611, USA.
authors.forename: Steven D
authors.initials: SD
authors.lastname: Bruner
authors.affiliation: Department of Medicinal Chemistry, Center for Natural Products, Drug Discovery and Development, College of Pharmacy, University of Florida, Gainesville, Florida, 32610, USA. yding@cop.ufl.edu.
authors.forename: Yousong
authors.initials: Y
authors.lastname: Ding