A Ribonucleotide Reductase‐Like Electron Transfer System in the Nitroaryl‐Forming N‐Oxygenase AurF

Fries, Alexander; Bretschneider, Tom; Winkler, Robert; Hertweck, Christian

doi:10.1002/cbic.201100138

Cited by 13 publications

(13 citation statements)

References 33 publications

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“…It has been proposed that the additional His residue stabilizes a diferric peroxo intermediate, which reacts directly with anilines rather than breaking down to higher valent iron species as in some di-iron proteins. 38,42,43 In conclusion, the differences in active site ligation between 3His-G4DFsc and G4DFsc indicate a direct relationship between active site structure and these differing reactivities. The parallels between the reactivities of these designed proteins and their natural counterparts are striking and imply that simplified, model proteins can be effective tools for studying and understanding the structure/function relationships in natural enzymes.…”

Section: Discussionmentioning

confidence: 77%

“…Genes were sequenced (UPenn Cell Center) following TAQ amplification to confirm the incorporation of the correct mutations and then transformed and expressed in E. coli BL21(DE3) cells (Novagen). Cells were lysed using the freeze-thaw method 43 and the crude lysate was collected via centrifugation at 15,000 rpm for 20 minutes. The protein was purified and reconstituted as described in the Supplementary Methods.…”

Section: Methodsmentioning

confidence: 99%

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Alteration of the oxygen-dependent reactivity of de novo Due Ferri proteins

et al. 2012

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De novo proteins provide a unique opportunity for investigating the structure-function relationships of metalloproteins in a minimal, well-defined, and controlled scaffold. Herein, we describe the rational programming of function in a de novo designed di-iron carboxylate protein from the due ferri family. Originally created to catalyze O2-dependent, two-electron oxidation of hydroquinones, the protein was reprogrammed to catalyze the selective N-hydroxylation of arylamines by remodeling the substrate access cavity and introducing a critical third His ligand to the metal binding cavity. Additional second-and third-shell modifications were required to stabilize the His ligand in the core of the protein. These changes resulted in at least a 106 –fold increase in the relative rates of the two reactions. This result highlights the potential for using de novo proteins as scaffolds for future investigations of geometric and electronic factors that influence the catalytic tuning of di-iron active sites.

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Section: Discussionmentioning

confidence: 77%

Section: Methodsmentioning

confidence: 99%

Alteration of the oxygen-dependent reactivity of de novo Due Ferri proteins

et al. 2012

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“…16,17 It has been noted that the AurF can be reconstituted in the heterologous host due to a ribonucleotide reductase-like electron transfer pathway, and CmlI likely works by a similar mechanism by using reduced ferredoxins as a source for electrons. 8 This is supported by the strict conservation of residues involved in electron transfer for CmlI and AurF (Fig. S1).…”

Section: Resultsmentioning

confidence: 66%

“…This is similar to what has been found for AurF, but as CmlI can accept much larger substrates, it may be more amenable for engineering as an N-oxidation catalyst. 8 It remains to be seen what other factors determine the substrate specificity of CmlI.…”

Section: Resultsmentioning

confidence: 99%

CmlI is an N-oxygenase in the biosynthesis of chloramphenicol

Chanco

Zhao

2012

Tetrahedron

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The N-oxygenation of an amine group is one of the steps in the biosynthesis of the antibiotic chloramphenicol. The non-heme di-iron enzyme CmlI was identified as the enzyme catalyzing this reaction through bioinformatics studies and reconstitution of enzymatic activity. In vitro reconstitution was achieved using phenazine methosulfate and NADH as electron mediators, while in vivo activity was demonstrated in Escherichia coli using two substrates. Kinetic analysis showed a biphasic behavior of the enzyme. Oxidized hydroxylamine and nitroso compounds in the reaction were detected both in vitro and in vivo based on LC–MS. The active site metal was confirmed to be iron based on a ferrozine assay. These findings provide new insights into the biosynthesis of chloramphenicol and could lead to further development of CmlI as a useful biocatalyst.

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“…251,262–271 In particular, the nature of the binuclear metallocofactor and the mechanism of oxidation were debated. An initial analysis of the AurF sequence revealed two copies of an EX 28–37 DEXXH motif that is conserved among several non-heme di-iron oxygenases.…”

Section: N–o Bond Forming Enzymesmentioning

confidence: 99%

Heteroatom–Heteroatom Bond Formation in Natural Product Biosynthesis

et al. 2017

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Natural products that contain functional groups with heteroatom-heteroatom linkages (X–X, where X = N, O, S, and P) are a small yet intriguing group of metabolites. The reactivity and diversity of these structural motifs has captured the interest of synthetic and biological chemists alike. Functional groups containing X–X bonds are found in all major classes of natural products and often impart significant biological activity. This review presents our current understanding of the biosynthetic logic and enzymatic chemistry involved in the construction of X–X bond containing functional groups within natural products. Elucidating and characterizing biosynthetic pathways that generate X–X bonds could both provide tools for biocatalysis and synthetic biology, as well as guide efforts to uncover new natural products containing these structural features.

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A Ribonucleotide Reductase‐Like Electron Transfer System in the Nitroaryl‐Forming N‐Oxygenase AurF

Cited by 13 publications

References 33 publications

Alteration of the oxygen-dependent reactivity of de novo Due Ferri proteins

Alteration of the oxygen-dependent reactivity of de novo Due Ferri proteins

CmlI is an N-oxygenase in the biosynthesis of chloramphenicol

Heteroatom–Heteroatom Bond Formation in Natural Product Biosynthesis

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