Structural Insights into the Mechanism of Base Excision by MBD4

DNA glycosylases remove damaged or modified nucleobases by cleaving the N-glycosyl bond and the correct nucleotide is restored through subsequent base excision repair. In addition to excising threatening lesions, DNA glycosylases contribute to epigenetic regulation by mediating DNA demethylation and perform other important functions. However, the catalytic mechanism remains poorly defined for many glycosylases, including MBD4 (methyl-CpG binding domain IV), a member of the helix-hairpin-helix (HhH) superfamily. MBD4 excises thymine from G·T mispairs, suppressing mutations caused by deamination of 5-methylcytosine, and it removes uracil and modified uracils (e.g., 5-hydroxymethyluracil) mispaired with guanine. To investigate the mechanism of MBD4 we solved high-resolution structures of enzyme-DNA complexes at three stages of catalysis. Using a non-cleavable substrate analog, 2′-deoxy-pseudouridine, we determined the first structure of an enzyme-substrate complex for wild-type MBD4, which confirms interactions that mediate lesion recognition and suggests that a catalytic Asp, highly conserved in HhH enzymes, binds the putative nucleophilic water molecule and stabilizes the transition state. Observation that mutating the Asp (to Gly) reduces activity by 2700-fold indicates an important role in catalysis, but probably not one as the nucleophile in a double-displacement reaction, as previously suggested. Consistent with direct-displacement hydrolysis, a structure of the enzyme-product complex indicates a reaction leading to inversion of configuration. A structure with DNA containing 1-azadeoxyribose models a potential oxacarbenium-ion intermediate and suggests the Asp could facilitate migration of the electrophile towards the nucleophilic water. Finally, the structures provide detailed snapshots of the HhH motif, informing how these ubiquitous metal-binding elements mediate DNA binding.     

Enrichment and characterization of ammonia-oxidizing archaea from the open ocean: phylogeny,physiology and stable isotope fractionation

Archaeal genes for ammonia oxidation are widespread in the marine environment, but direct physiological evidence for ammonia oxidation by marine archaea is limited. We report the enrichment and characterization of three strains of pelagic ammonia-oxidizing archaea (AOA) from the North Pacific Ocean that have been maintained in laboratory culture for over 3 years. Phylogenetic analyses indicate the three strains belong to a previously identified clade of water column-associated AOA and possess 16S ribosomal RNA genes and ammonia monooxygenase subunit a (amoA) genes highly similar (98–99% identity) to those recovered in DNA and complementary DNA clone libraries from the open ocean. The strains grow in natural seawater-based liquid medium while stoichiometrically converting ammonia (NH₃) to nitrite (NO₂⁻). Ammonia oxidation by the enrichments is only partially inhibited by allylthiourea at concentrations known to completely inhibit cultivated ammonia-oxidizing bacteria. The three strains were used to determine the nitrogen stable isotope effect (¹⁵ɛ_NH3) during archaeal ammonia oxidation, an important parameter for interpreting stable isotope ratios in the environment. Archaeal ¹⁵ɛ_NH3 ranged from 13‰ to 41‰, within the range of that previously reported for ammonia-oxidizing bacteria. Despite low amino acid identity between the archaeal and bacterial Amo proteins, their functional diversity as captured by ¹⁵ɛ_NH3 is similar.     

Host identification in unfed ticks from stable isotope compositions (δ13C and δ15N)

Determination of the ratios of natural stable isotopes (¹³C/¹²C and ¹⁵N/¹⁴N) in unfed Ixodes ricinus nymphs and adults, which, in their previous stage, fed on captive wild rodents (Apodemus sylvaticus and Myodes glareolus), wild birds (Parus major and Cyanistes caeruleus) or domestic ruminants (Ovis aries and Bos taurus), demonstrated that it is possible to identify each host category with confidence. First, the tick–blood spacing, which is the difference between values obtained from ticks and the blood of hosts that they had fed on in the previous stage, was consistent (152 spacings investigated from 15 host individuals in total). Second, potential confounding factors (tick age and sex) did not affect the discriminatory power of the isotope patterns, nor did different rearing conditions (room temperature vs. 4 °C) or the duration of development (maximum of 430 days). The findings that the tick–blood isotope spacings, across a diverse range of hosts, were similar and predictable, and that confounders had little or no effect on this, strongly support the usage of the isotope approach. Because each of the host categories has a different role in the population dynamics of I. ricinus and in tick‐borne pathogen ecology, the method described here has great potential for the clarification of tick and tick‐borne pathogen ecology in the field.     

Direct anabolic metabolism of three-carbon propionate to a six-carbon metabolite occurs in vivo across tissues and species

Anabolic metabolism of carbon in mammals is mediated via the one- and two-carbon carriers S-adenosyl methionine and acetyl-coenzyme A. In contrast, anabolic metabolism of three-carbon units via propionate has not been shown to extensively occur. Mammals are primarily thought to oxidize the three-carbon short chain fatty acid propionate by shunting propionyl-CoA to succinyl-CoA for entry into the TCA cycle. Here, we found that this may not be absolute as, in mammals, one nonoxidative fate of propionyl-CoA is to condense to two three-carbon units into a six-carbon trans-2-methyl-2-pentenoyl-CoA (2M2PE-CoA). We confirmed this reaction pathway using purified protein extracts provided limited substrates and verified the product via LC-MS using a synthetic standard. In whole-body in vivo stable isotope tracing following infusion of ¹³C-labeled valine at steady state, 2M2PE-CoA was found to form via propionyl-CoA in multiple murine tissues, including heart, kidney, and to a lesser degree, in brown adipose tissue, liver, and tibialis anterior muscle. Using ex vivo isotope tracing, we found that 2M2PE-CoA also formed in human myocardial tissue incubated with propionate to a limited extent. While the complete enzymology of this pathway remains to be elucidated, these results confirm the in vivo existence of at least one anabolic three- to six-carbon reaction conserved in humans and mice that utilizes propionate.     

Biological nitrogen fixation in grass–clover affected by animal excreta

Aiming at estimating the average N2-fixation in a pasture, ap preciating the great variability due to patchy urine and dung deposition, the in fluence of dairy cow excreta on biological N2-fixation in a perennial ryegrass–white clover mixture was studied using natural urine and dung. Application of urine as well as dung affected the N2-fixation by promoting the growth of grass and thereby the proportion of clover was significantly reduced. Also the proportion of clover-N derived from the atmosphere (pNdfa) was significantly reduced. In control plots clover dry matter constituted between 40 and 50% of the total dry matter production and the pNdfa ranged between 0.8 and 0.9. Addition of urine caused a significant increase in the grass growth rates, which was the primary reason for a decrease in proportion of clover. At the same time pNdfa decreased to 0.2–0.4 followed by an increase resulting in a total reduction of 45% in the N2-fixation in urine affected areas over a period of four months. The dung only affected the N2-fixation for a distance of up to 10 cm from the edge of the dung pats. In this border area the pNdfa decreased from 0.85 to 0.75 during one month after application followed by an increase, so that after three months there was no difference between pNdfa at 0–10 and 10–20 cm distance from the dung hill. The proportion of clover was lower in the 0–10 cm than in the 10–20 cm distance, which totally resulted in a total reduction of 20% in the N2-fixation over a period of four months in the 0–10 cm area around the dung pats. Considering the proportion of a pasture which may by affected by excreta at a stocking density of 4–6 cows ha-1, the length of the grazing period, the frequency of excretion and the area covered by individual patches, it was estimated that the N2-fixation in a grass-clover pasture would be reduced by 10–15% compared to the N2-fixation in a grass-clover sward not exposed to animal excreta.     

Regional differences in the carbon isotopic compositions of teak from two monsoonal regimes of India

Inter and intra-annual carbon isotope compositions (δ¹³C) of several annual growth rings of teak trees from two monsoonal regimes from India were studied and compared with the corresponding oxygen isotopic (δ¹⁸O) variations. In teak from both the regimes, amplitudes of intra-annual δ¹³C were ∼2-3 times lower than that observed in δ¹⁸O. Seasonal cycle with lower δ¹³C values at the middle and higher at ring boundaries was observed for teak from central India, dominated by the southwest monsoon. Positive correlations of intra-annual δ¹³C values with the corresponding δ¹⁸O values of the same rings and with relative humidity (RH) of the concurrent period suggest a dominant role of RH in controlling δ¹³C values of teak from central India. Intra-annual δ¹³C variations of teak from southern India, receiving both the southwest and northeast monsoons, revealed an initial decreasing trend followed by an increasing trend before culminating in depleted ¹³C values at the end of the growing season. No correlation was observed between intra-annual δ¹³C and δ¹⁸O variations of teak trees from southern India. Regional differences in the climatology of δ¹³C of atmospheric CO₂ or the lengths of growing season could be likely reasons for differing intra-annual δ¹³C variations of teak from the two climatic regimes.