A novel alkane monooxygenase evolved from a broken piece of ribonucleotide reductase in Geobacillus kaustophilus HTA426 isolated from Mariana Trench.

We have accidentally found that a thermophilic Geobacillus kaustophilus HTA426 is capable of degrading alkanes although it has no alkane oxygenating enzyme genes. Our experimental results revealed that a putative ribonucleotide reductase small subunit GkR2loxI (GK2771) gene encodes a novel heterodinuclear Mn-Fe alkane monooxygenase/hydroxylase. GkR2loxI protein can perform two-electron oxidations similar to homonuclear diiron bacterial multicomponent soluble methane monooxygenases. This finding not only answers a long-standing question about the substrate of the R2lox protein clade, but also expands our understanding of the vast diversity and new evolutionary lineage of the bacterial alkane monooxygenase/hydroxylase family.

Reflections on Charlie Gard and the Best Interests Standard From Both Sides of the Atlantic Ocean.

Charlie Gard (August 4, 2016, to July 28, 2017) was an infant in the United Kingdom who was diagnosed with an encephalopathic form of mitochondrial DNA depletion syndrome caused by a mutation in the RRM2B gene. Charlie's parents raised £1.3 million (∼$1.6 million US) on a crowdfunding platform to travel to New York to pursue experimental nucleoside bypass treatment, which was being used to treat a myopathic form of mitochondrial DNA depletion syndrome caused by mutations in a different gene (TK2). The case made international headlines about what was in Charlie's best interest. In the medical ethics community, it raised the question of whether best interest serves as a guidance principle (a principle that provides substantive directions as to how decisions are to be made), an intervention principle (a principle specifying the conditions under which third parties are to intervene), both guidance and intervention, or neither. I show that the United Kingdom uses best interest as both guidance and intervention, and the United States uses best interest for neither. This explains why the decision to withdraw the ventilator without attempting nucleoside bypass treatment was the correct decision in the United Kingdom and why the opposite conclusion would have been reached in the United States.

Genotoxicity of chemical compounds identification and assessment by yeast cells transformed with GFP reporter constructs regulated by the PLM2 or DIN7 promoter.

Yeast cells transformed with high-copy number plasmids comprising a green fluorescent protein (GFP)-encoding gene optimized for yeast under the control of the new DIN7 or PLM2 and the established RNR2 and RAD54 promoters were used to assess the genotoxic potential of chemical compounds. The activity of potential DNA-damaging agents was investigated by genotoxicity assays and by OxoPlate assay in the presence of various test compounds. The fluorescence signal generated by GFP in response to DNA damage was related to the different concentrations of analytes and the analyte-dependent GFP synthesis. The use of distinct DNA damage-inducible promoters presents alternative genotoxicity testing strategies by selective induction of promoters in response to DNA damage. The new DIN7 and PLM2 systems show higher sensitivity than the RNR2 and RAD54 systems in detecting 4-nitroquinoline-N-oxide and actinomycin D. Both DIN7 and PLM2 systems are able to detect camptothecin while RNR2 and RAD54 systems are not. Automated laboratory systems with assay performance on 384-well microplates provide for cost-effective high-throughput screening of DNA-damaging agents, reducing compound consumption to about 53% as compared with existing eukaryotic genotoxicity bioassays.

A genotoxicity test system based on p53R2 gene expression in human cells: assessment of its reactivity to various classes of genotoxic chemicals.

The tumor suppressor, p53, plays an important role in DNA damage repair, by regulating the expression of target genes. One p53-target gene, p53R2, which encodes a subunit of ribonucleotide reductase, is activated by DNA damage. We have previously developed a genotoxicity test system, using human cell lines and a p53R2-dependent luciferase reporter gene assay. 80 chemicals have been examined with this system and 40 of 43 Ames-positive chemicals induced luciferase activity. Eight Ames-negative genotoxic chemicals also induced luciferase activity. Although this assay system could, potentially, be applied to the rapid screening of chemicals that are potentially genotoxic to humans, the ability of the assay to detect genotoxic effects was unclear. In this study, to evaluate the performance of this assay system, several different types of DNA damaging agents were screened. 27 chemicals, whose genotoxic mechanisms are well known, were screened. All genotoxic compounds, except for anti-metabolites and histone deacetylase HDAC inhibitors, showed significant luciferase activity with the following rank order of potency: topoisomerase II inhibitors, intercalaters>bleomycin>topoisomerase I inhibitors>alkylating agents=DNA cross-linking agents=polycyclic aromatic hydrocarbons>spindle poisons. This assay showed greater response to those genotoxic agents that induce DNA double strand break damage compared to those agents that cause other forms of DNA damage. DNA double strand breakage initiates genomic instability, a feature of carcinogenicity. These results indicate that this assay system could be a helpful tool for predicting chemical genotoxicity and carcinogenicity in humans.

New paramagnetic species formed at the expense of the transient tyrosyl radical in mutant protein R2 F208Y of Escherichia coli ribonucleotide reductase.

The highly conserved residue F208 in protein R2 of E. coli ribonucleotide reductase is close to the binuclear iron center, and found to be involved in stabilizing the tyrosyl radical Y122. in wild type R2. Upon the reconstitution reaction of the mutant R2 F208Y with ferrous iron and molecular oxygen, we observed a new EPR singlet signal (g = 2.003) formed concomitantly with decay of the transient tyrosyl radical Y122. (g = 2.005). This new paramagnetic species (denoted Z) was stable for weeks at 4 degrees C and visible by EPR only below 50 K. The EPR singlet could not be saturated by available microwave power, suggesting that Z may be a mainly metal centered species. The maximum amount of the compound Z in the protein purified from cells grown in rich medium was about 0.18 unpaired spin/R2. An identical EPR signal of Z was found also in the double mutant R2 F208Y/Y122F. In the presence of high concentration of sodium ascorbate, the amounts of both the transient Y122. and the new species Z increased considerably in the reconstitution reaction. The results suggest that Z is most likely an oxo-ferryl species possibly in equilibrium with a Y208 ligand radical.

Molecular characterization of a ribonucleotide reductase (nrdF) gene fragment of Mycoplasma hyopneumoniae and assessment of the recombinant product as an experimental vaccine for enzootic pneumonia.

A Mycoplasma hyopneumoniae clone bank was screened with hyperimmune pig serum. One clone exhibited sequence homology to the prokaryotic R2 subunit of ribonucleotide reductase and was expressed as an 11-kDa protein fused to beta-galactosidase. The vaccine potential of the fusion protein was assessed in pig trials. Following experimental challenge with a virulent isolate of M. hyopneumoniae, gross lung pathology (mean Goodwin lung score) of vaccinated animals, irrespective of adjuvant treatment, was significantly reduced compared with that of control unvaccinated pigs (P < 0.05).