DNA building blocks: keeping control of manufacture

Ribonucleotide reductase (RNR) is the only source for de novo production of the four deoxyribonucleoside triphosphate (dNTP) building blocks needed for DNA synthesis and repair. It is crucial that these dNTP pools are carefully balanced, since mutation rates increase when dNTP levels are either unbalanced or elevated. RNR is the major player in this homeostasis, and with its four different substrates, four different allosteric effectors and two different effector binding sites, it has one of the most sophisticated allosteric regulations known today. In the past few years, the structures of RNRs from several bacteria, yeast and man have been determined in the presence of allosteric effectors and substrates, revealing new information about the mechanisms behind the allosteric regulation. A common theme for all studied RNRs is a flexible loop that mediates modulatory effects from the allosteric specificity site (s-site) to the catalytic site for discrimination between the four substrates. Much less is known about the allosteric activity site (a-site), which functions as an on-off switch for the enzyme's overall activity by binding ATP (activator) or dATP (inhibitor). The two nucleotides induce formation of different enzyme oligomers, and a recent structure of a dATP-inhibited α6β2 complex from yeast suggested how its subunits interacted non-productively. Interestingly, the oligomers formed and the details of their allosteric regulation differ between eukaryotes and Escherichia coli Nevertheless, these differences serve a common purpose in an essential enzyme whose allosteric regulation might date back to the era when the molecular mechanisms behind the central dogma evolved

Spectroscopic Studies of the Iron and Manganese Reconstituted Tyrosyl Radical in Bacillus Cereus Ribonucleotide Reductase R2 Protein

Ribonucleotide reductase (RNR) catalyzes the rate limiting step in DNA synthesis where ribonucleotides are reduced to the corresponding deoxyribonucleotides. Class Ib RNRs consist of two homodimeric subunits: R1E, which houses the active site; and R2F, which contains a metallo cofactor and a tyrosyl radical that initiates the ribonucleotide reduction reaction. We studied the R2F subunit of B. cereus reconstituted with iron or alternatively with manganese ions, then subsequently reacted with molecular oxygen to generate two tyrosyl-radicals. The two similar X-band EPR spectra did not change significantly over 4 to 50 K. From the 285 GHz EPR spectrum of the iron form, a g1-value of 2.0090 for the tyrosyl radical was extracted. This g1-value is similar to that observed in class Ia E. coli R2 and class Ib R2Fs with iron-oxygen cluster, suggesting the absence of hydrogen bond to the phenoxyl group. This was confirmed by resonance Raman spectroscopy, where the stretching vibration associated to the radical (C-O, ν7a = 1500 cm−1) was found to be insensitive to deuterium-oxide exchange. Additionally, the 18O-sensitive Fe-O-Fe symmetric stretching (483 cm−1) of the metallo-cofactor was also insensitive to deuterium-oxide exchange indicating no hydrogen bonding to the di-iron-oxygen cluster, and thus, different from mouse R2 with a hydrogen bonded cluster. The HF-EPR spectrum of the manganese reconstituted RNR R2F gave a g1-value of ∼2.0094. The tyrosyl radical microwave power saturation behavior of the iron-oxygen cluster form was as observed in class Ia R2, with diamagnetic di-ferric cluster ground state, while the properties of the manganese reconstituted form indicated a magnetic ground state of the manganese-cluster. The recent activity measurements (Crona et al., (2011) J Biol Chem 286: 33053–33060) indicates that both the manganese and iron reconstituted RNR R2F could be functional. The manganese form might be very important, as it has 8 times higher activity

Global Distribution of Outbreaks of Water-Associated Infectious Diseases

Water is essential for maintaining life on Earth but can also serve as a media for many pathogenic organisms, causing a high disease burden globally. However, how the global distribution of water-associated infectious pathogens/diseases looks like and how such distribution is related to possible social and environmental factors remain largely unknown. In this study, we compiled a database on distribution, biology, and epidemiology of water-associated infectious diseases and collected data on population density, annual accumulated temperature, surface water areas, average annual precipitation, and per capita GDP at the global scale. From the database we extracted reported outbreak events from 1991 to 2008 and developed models to explore the association between the distribution of these outbreaks and social and environmental factors. A total of1,428 outbreaks had been reported and this number only reflected ‘the tip of the iceberg’ of the much bigger problem. We found that the outbreaks of water-associated infectious diseases are significantly correlated with social and environmental factors and that all regions are affected disproportionately by different categories of diseases. Relative risk maps are generated to show ‘hotspots’ of risks for different diseases. Despite certain limitations, the findings may be instrumental for future studies and prioritizing health resources

Pathogenic mycobacteria in water

The book concludes with a discussion of the issues surrounding the control of PEM indrinking water and the assessment and management of risks