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Abstract Mononuclear nonheme iron(II) and 2‐oxoglutarate (Fe/2OG)‐dependent oxygenases and halogenases are known to catalyze a diverse set of oxidative reactions, including hydroxylation, halogenation, epoxidation, and desaturation in primary metabolism and natural product maturation. However, their use in abiotic transformations has mainly been limited to C−H oxidation. Herein, we show that various enzymes of this family, when reconstituted with Fe(II) or Fe(III), can catalyze Mukaiyama hydration—a redox neutral transformation. Distinct from the native reactions of the Fe/2OG enzymes, wherein oxygen atom transfer (OAT) catalyzed by an iron‐oxo species is involved, this nonnative transformation proceeds through a hydrogen atom transfer (HAT) pathway in a 2OG‐independent manner. Additionally, in contrast to conventional inorganic catalysts, wherein a dinuclear iron species is responsible for HAT, the Fe/2OG enzymes exploit a mononuclear iron center to support this reaction. Collectively, our work demonstrates that Fe/2OG enzymes have utility in catalysis beyond the current scope of catalytic oxidation.
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Engineering sequence and selectivity of late-stage C-H oxidation in the MycG iterative cytochrome P450
Abstract MycG is a multifunctional P450 monooxygenase that catalyzes sequential hydroxylation and epoxidation or a single epoxidation in mycinamicin biosynthesis. In the mycinamicin-producing strain Micromonospora griseorubida A11725, very low-level accumulation of mycinamicin V generated by the initial C-14 allylic hydroxylation of MycG is observed due to its subsequent epoxidation to generate mycinamicin II, the terminal metabolite in this pathway. Herein, we investigated whether MycG can be engineered for production of the mycinamicin II intermediate as the predominant metabolite. Thus, mycG was subject to random mutagenesis and screening was conducted in Escherichia coli whole-cell assays. This enabled efficient identification of amino acid residues involved in reaction profile alterations, which included MycG R111Q/V358L, W44R, and V135G/E355K with enhanced monohydroxylation to accumulate mycinamicin V. The MycG V135G/E355K mutant generated 40-fold higher levels of mycinamicin V compared to wild-type M. griseorubida A11725. In addition, the E355K mutation showed improved ability to catalyze sequential hydroxylation and epoxidation with minimal mono-epoxidation product mycinamicin I compared to the wild-type enzyme. These approaches demonstrate the ability to selectively coordinate the catalytic activity of multifunctional P450s and efficiently produce the desired compounds.
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- Award ID(s):
- 1700982
- PAR ID:
- 10367172
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Journal of Industrial Microbiology and Biotechnology
- Volume:
- 49
- Issue:
- 1
- ISSN:
- 1367-5435
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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