Thaxtomin phytotoxins produced by plant-pathogenic Streptomyces species contain a nitro group that is essential for phytotoxicity. The N,N’-dimethyldiketopiperazine core of thaxtomins is assembled from L-phenylalanine and L-4-nitrotryptophan by a nonribosomal peptide synthetase and nitric oxide synthase-generated NO is incorporated into the nitro group, but the biosynthesis of the non-proteinogenic amino acid L-4-nitrotryptophan is unclear. Here we report that TxtE, a unique cytochrome P450, catalyzes L-tryptophan nitration using NO and O2.

Rieske non-heme iron-dependent oxygenases are important enzymes that catalyze a wide variety of reactions in the biodegradation of xenobiotics and the biosynthesis of bioactive natural products. In this perspective article, we summarize recent efforts to elucidate the catalytic mechanisms of Rieske oxygenases and highlight the diverse range of reactions now known to be catalyzed by such enzymes.

Oxidative cyclizations, exemplified by the biosynthetic assembly of the penicillin nucleus from a tripeptide precursor, are arguably the most synthetically-powerful implementation of C-H activation reactions in Nature. Here we show that Rieske oxygenase-like enzymes mediate regio and stereodivergent oxidative cyclizations to form 10- and 12-membered carbocyclic rings in the key steps of the biosynthesis of the antibiotics streptorubin B and metacycloprodigiosin, respectively. These reactions represent the first examples of oxidative carbocyclizations catalyzed by non-heme iron-dependent oxidases and define a novel type of catalytic activity for Rieske enzymes. A better understanding of how these enzymes achieve such remarkable regio and stereocontrol in the functionalization of unactivated hydrocarbon chains will greatly facilitate the development of selective manmade C-H activation catalysts.

Gliotoxin, a redox-active metabolite, is produced by the opportunistic fungal pathogen Aspergillus fumigatus, and its biosynthesis is directed by the gli gene cluster. Knowledge of the biosynthetic pathway to gliotoxin, which contains a disulfide bridge of unknown origin, is limited, although L-Phe and L-Ser are known biosynthetic precursors. Deletion of gliG from the gli cluster, herein functionally confirmed as a glutathione S-transferase, results in abrogation of gliotoxin biosynthesis and accumulation of 6-benzyl-6-hydroxy-1-methoxy-3-methylenepiperazine-2,5-dione. This putative shunt metabolite from the gliotoxin biosynthetic pathway contains an intriguing hydroxyl group at C-6, consistent with a gliotoxin biosynthetic pathway involving thiolation via addition of the glutathione thiol group to a reactive acyl imine intermediate. Complementation of gliG restored gliotoxin production and, unlike gliT, gliG was found not to be involved in fungal self-protection against gliotoxin.

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The cytochromes P450 are heme-dependent enzymes that catalyze many vital reaction processes in the human body related to biodegradation and biosynthesis. They typically act as mono-oxygenases; however, the recently discovered P450 subfamily TxtE utilizes O 2 and NO to nitrate aromatic substrates such as L -tryptophan. A direct and selective aromatic nitration reaction may be useful in biotechnology for the synthesis of drugs or small molecules. Details of the catalytic mechanism are unknown, and it has been suggested that the reaction should proceed through either an iron(III)-superoxo or an iron(II)-nitrosyl intermediate. To resolve this controversy, we used stopped-flow kinetics to provide evidence for a catalytic cycle where dioxygen binds prior to NO to generate an active iron(III)-peroxynitrite species that is able to nitrate l -Trp efficiently. We show that the rate of binding of O 2 is faster than that of NO and also leads to l -Trp nitration, while little evidence of product formation is observed from the iron(II)-nitrosyl complex. To support the experimental studies, we performed density functional theory studies on large active site cluster models. The studies suggest a mechanism involving an iron(III)-peroxynitrite that splits homolytically to form an iron(IV)-oxo heme (Compound II) and a free NO 2 radical via a small free energy of activation. The latter activates the substrate on the aromatic ring, while compound II picks up the ipso -hydrogen to form the product. The calculations give small reaction barriers for most steps in the catalytic cycle and, therefore, predict fast product formation from the iron(III)-peroxynitrite complex. These findings provide the first detailed insight into the mechanism of nitration by a member of the TxtE subfamily and highlight how the enzyme facilitates this novel reaction chemistry.

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Iron is emerging as a key player in the search for efficient and environmentally benign methods for the functionalisation of C-H bonds. Non-heme iron enzymes catalyse a diverse array of oxidative chemistry in nature, and small-molecule complexes designed to mimic the non-heme iron active site have great potential as C-H activation catalysts. Herein we report the synthesis of a series of organic ligands that incorporate key features of the non-heme iron active site. Iron(II) complexes of these ligands have been generated in situ and their ability to promote hydrocarbon oxidation has been investigated. Several of these systems promote the biomimetic dihydroxylation of cyclohexene at low levels, when hydrogen peroxide is used as the oxidant; allylic oxidation products are also observed. An investigation of ligand stability reveals formation of several breakdown products under the conditions of the oxidative turnover reactions. These products arise via oxidative decarboxylation, dehydration and deamination reactions. Taken together these results indicate that competing mechanisms are at play with these systems: biomimetic hydroxylation involving high-valent iron species, and allylic oxidation via Fenton chemistry and Haber-Weiss radical pathways.