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.
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.
more » « less- Award ID(s):
- 1700982
- NSF-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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Abstract -
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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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Abstract Background Advances in microbiome science are being driven in large part due to our ability to study and infer microbial ecology from genomes reconstructed from mixed microbial communities using metagenomics and single-cell genomics. Such omics-based techniques allow us to read genomic blueprints of microorganisms, decipher their functional capacities and activities, and reconstruct their roles in biogeochemical processes. Currently available tools for analyses of genomic data can annotate and depict metabolic functions to some extent; however, no standardized approaches are currently available for the comprehensive characterization of metabolic predictions, metabolite exchanges, microbial interactions, and microbial contributions to biogeochemical cycling.
Results We present METABOLIC (METabolic And BiogeOchemistry anaLyses In miCrobes), a scalable software to advance microbial ecology and biogeochemistry studies using genomes at the resolution of individual organisms and/or microbial communities. The genome-scale workflow includes annotation of microbial genomes, motif validation of biochemically validated conserved protein residues, metabolic pathway analyses, and calculation of contributions to individual biogeochemical transformations and cycles. The community-scale workflow supplements genome-scale analyses with determination of genome abundance in the microbiome, potential microbial metabolic handoffs and metabolite exchange, reconstruction of functional networks, and determination of microbial contributions to biogeochemical cycles. METABOLIC can take input genomes from isolates, metagenome-assembled genomes, or single-cell genomes. Results are presented in the form of tables for metabolism and a variety of visualizations including biogeochemical cycling potential, representation of sequential metabolic transformations, community-scale microbial functional networks using a newly defined metric “MW-score” (metabolic weight score), and metabolic Sankey diagrams. METABOLIC takes ~ 3 h with 40 CPU threads to process ~ 100 genomes and corresponding metagenomic reads within which the most compute-demanding part of hmmsearch takes ~ 45 min, while it takes ~ 5 h to complete hmmsearch for ~ 3600 genomes. Tests of accuracy, robustness, and consistency suggest METABOLIC provides better performance compared to other software and online servers. To highlight the utility and versatility of METABOLIC, we demonstrate its capabilities on diverse metagenomic datasets from the marine subsurface, terrestrial subsurface, meadow soil, deep sea, freshwater lakes, wastewater, and the human gut.
Conclusion METABOLIC enables the consistent and reproducible study of microbial community ecology and biogeochemistry using a foundation of genome-informed microbial metabolism, and will advance the integration of uncultivated organisms into metabolic and biogeochemical models. METABOLIC is written in Perl and R and is freely available under GPLv3 at
https://github.com/AnantharamanLab/METABOLIC . -
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Abstract Mice missing either Reelin or Disabled‐1 (Dab1) exhibit dorsal horn neuronal positioning errors and display heat hypersensitivity and mechanical insensitivity. Reelin binds its receptors, apolipoprotein E receptor 2 and very low‐density lipoprotein receptor, leading to the recruitment and phosphorylation of Dab1 and activation of downstream pathways that regulate neuronal migration. Previously, we reported that 70% of Dab1 laminae I–II neurons co‐expressed LIM‐homeobox transcription factor 1‐beta (Lmx1b). Here, we asked whether Reelin‐expressing dorsal horn neurons co‐express Lmx1b, are mispositioned in
dab1 mutants, and contribute to nociceptive abnormalities. About 90% of Reelin‐labeled neurons are Lmx1b‐positive in laminae I–II, confirming that most Reelin and Dab1 neurons are glutamatergic. We determined that Reelin‐Lmx1b and Dab1‐Lmx1b dorsal horn neurons are separate populations, and together, comprise 37% of Lmx1b‐positive cells within and above the Isolectin B4 (IB4) layer in wild‐type mice. Compared to wild‐type mice,dab1 mutants have a reduced area of laminae I–II outer (above the IB4 layer), more Reelin‐Lmx1b neurons within the IB4 layer, and fewer Reelin‐Lmx1b neurons within the lateral reticulated area of lamina V and lateral spinal nucleus. Interestingly, both Reelin‐ and Dab1‐labeled dorsal horn neurons sustain similar positioning errors in mutant mice. After noxious thermal and mechanical stimulation, Reelin, Lmx1b, and Reelin‐Lmx1b neurons expressed Fos in laminae I–II and the lateral reticulated area in wild‐type mice and, therefore, participate in nociceptive circuits. Together, our data suggest that disruption of the Reelin‐signaling pathway results in neuroanatomical abnormalities that contribute to the nociceptive changes that characterize these mutant mice. -
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ABSTRACT BACKGROUND AND PURPOSE Mutations in isocitrate dehydrogenase (IDH) have a direct effect on gliomagenesis. The purpose of this study is to quantify differences in brain metabolites due to IDH mutations.
METHODS Magnetic Resonance Spectroscopic Imaging (MRSI) was performed in 35 patients with gliomas of different grade and varied IDH mutation status. Volumes of interest (VOIs) for active tumor (tVOI), peritumoral area (pVOI), and contralateral normal‐appearing white matter (cVOI) were created. Metabolite ratios of Choline (Cho) to both
N ‐acetylaspartate (NAA) and Creatine (Cr) were estimated. Ratios of Glutamate/Glutamine complex (Glx) and myoinositol (mIno) to Cr were also quantified. General linear models (GLMs) were used to estimate the effects of IDH mutation on metabolite measures, with age, gender, and tumor grade used as covariates.RESULTS GLM analysis showed that maximum Cho/NAA and Cho/Cr in the tVOI were significantly (
P < .05) higher in IDH mutant lesions as compared to wild‐type. In the pVOI, mean Cho/Cr was found to be significantly different among IDH mutant and wild‐type gliomas. Mean Cho/NAA (P = .306) and Cho/Cr (P = .292) within the tVOI were not significantly different. Ratios of Glx/Cr and mIno/Cr in any region showed no significant differences between IDH mutant and wild‐type gliomas. No significant differences in metabolite ratios were seen in the cVOI between IDH mutants and wild‐types.CONCLUSION IDH mutation's effect in gliomas show an increase in Cho in the tumor and perilesional regions as compared to wild‐type lesions but do not show widespread changes across the brain.
