title: 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.
authors.affiliation: Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, Florida, USA.
authors.forename: Ran
authors.initials: R
authors.lastname: Zuo
authors.affiliation: Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, Florida, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Plant Pathology, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida, USA.
authors.forename: Jose C
authors.initials: JC
authors.lastname: Huguet-Tapia
authors.affiliation: Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, Florida, USA.
authors.forename: Mishal
authors.initials: M
authors.lastname: Mehta
authors.affiliation: Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, Florida, USA.
authors.forename: Evelina
authors.initials: E
authors.lastname: Dedic
authors.affiliation: Department of Chemistry, University of Florida, Gainesville, Florida, USA.|Center for Natural Products, Drug Discovery and Development, College of Pharmacy, University of Florida, Gainesville, Florida, USA.
authors.forename: Steven D
authors.initials: SD
authors.lastname: Bruner
authors.affiliation: Department of Plant Pathology, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida, USA.
authors.forename: Rosemary
authors.initials: R
authors.lastname: Loria
authors.affiliation: Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, Florida, USA. yding@cop.ufl.edu.|Center for Natural Products, Drug Discovery and Development, College of Pharmacy, University of Florida, Gainesville, Florida, USA. yding@cop.ufl.edu.
authors.forename: Yousong
authors.initials: Y
authors.lastname: Ding
mesh_terms: D001665:Binding Sites; D055162:Biocatalysis; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D005461:Fluorine / Q000737:chemistry*; D007211:Indoles / Q000737:chemistry; D009566:Nitrates / Q000737:chemistry*; D000072417:Protein Domains; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D014364:Tryptophan / Q000031:analogs & derivatives / Q000737:chemistry* / Q000302:isolation & purification*
chemical_list: D007211:Indoles; D009566:Nitrates; D011993:Recombinant Fusion Proteins; D005461:Fluorine; C030374:indole; D014364:Tryptophan; D003577:Cytochrome P-450 Enzyme System
keywords: Direct nitration; Fluorinated tryptophan; P450; Regio-selectivity; Self-sufficient