Anna Larsson

Structure and function of carbonic anhydrases from mycobacterium tuberculosis

Carbonic anhydrases catalyze the reversible hydration of carbon dioxide to form bicarbonate. This activity is universally required for fatty acid biosynthesis as well as for the production of a number of small molecules, pH homeostasis, and other functions. At least three different carbonic anhydrase families are known to exist, of which the α-class found in humans has been studied in most detail. In the present work, we describe the structures of two of the three β-class carbonic anhydrases that have been identified in Mycobacterium tuberculosis, i.e. Rv1284 and Rv3588c. Both structures were solved by molecular replacement and then refined to resolutions of 2.0 and 1.75 Å, respectively. The active site of Rv1284 is small and almost completely shielded from solvent, whereas that of Rv3588c is larger and quite open to solution. Differences in coordination of the active site metal are also observed. In Rv3588c, an aspartic acid side chain displaces a water molecule and coordinates directly to the zinc ion, thereby closing the zinc coordination sphere and breaking the salt link to a nearby arginine that is a feature of Rv1284. The two carbonic anhydrases thus exhibit both of the metal coordination geometries that have previously been observed for structures in this family. Activity studies demonstrate that Rv3588c is a completely functional carbonic anhydrase. The apparent lack of activity of Rv1284 in the present assay system is likely exacerbated by the observed depletion of zinc in the preparation.

Crystal Structure and Computational Characterization of the Lytic Polysaccharide Monooxygenase Gh61D from the Basidiomycota Fungus Phanerochaete Chrysosporium

Background: Lytic polysaccharide monooxygenases (LPMOs) represent a recently discovered enzymatic route to cleave carbohydrates. Results: We report the first basidiomycete LPMO structure and describe enzyme-cellulose interactions with simulation. Conclusion: We characterize the copper-containing active site and identify loops important for substrate recognition and binding. Significance: This structure is the first LPMO from a model basidiomycete fungus that contains many LPMO genes. Carbohydrate structures are modified and degraded in the biosphere by a myriad of mostly hydrolytic enzymes. Recently, lytic polysaccharide mono-oxygenases (LPMOs) were discovered as a new class of enzymes for cleavage of recalcitrant polysaccharides that instead employ an oxidative mechanism. LPMOs employ copper as the catalytic metal and are dependent on oxygen and reducing agents for activity. LPMOs are found in many fungi and bacteria, but to date no basidiomycete LPMO has been structurally characterized. Here we present the three-dimensional crystal structure of the basidiomycete Phanerochaete chrysosporium GH61D LPMO, and, for the first time, measure the product distribution of LPMO action on a lignocellulosic substrate. The structure reveals a copper-bound active site common to LPMOs, a collection of aromatic and polar residues near the binding surface that may be responsible for regio-selectivity, and substantial differences in loop structures near the binding face compared with other LPMO structures. The activity assays indicate that this LPMO primarily produces aldonic acids. Last, molecular simulations reveal conformational changes, including the binding of several regions to the cellulose surface, leading to alignment of three tyrosine residues on the binding face of the enzyme with individual cellulose chains, similar to what has been observed for family 1 carbohydrate-binding modules. A calculated potential energy surface for surface translation indicates that P. chrysosporium GH61D exhibits energy wells whose spacing seems adapted to the spacing of cellobiose units along a cellulose chain.

Assessing the feasibility of targeted selective treatments for gastrointestinal nematodes in first-season grazing cattle based on mid-season daily weight gains

A retrospective study was carried out to assess the possibility of using daily weight gain in first-season grazing cattle (FSG) as a marker for treatment decisions to prevent parasite-induced losses caused by gastrointestinal (GI) nematodes. This is intended as a proof of concept for using targeted selective treatments (TSTs) in the control of veterinary parasites. Historical data were combined from three independent grazing trials in Sweden, each of which was repeated over 2-3 years, in order to investigate the influences of parasites on the performance of FSG cattle subjected to different levels of parasite control. In general, the different trials and years produced congruent results. There was a good positive linear relationship between daily weight gains (Dwgt) registered around housing and those recorded in the middle of the grazing season (r(2)=0.54) but not between early-season Dwgt and housing Dwgt. The latter observation emphasizes that factors other than nematode infection influence the final weight of the cattle. However, significant differences in Dwgt ( approximately 0.2 kg/day) were also observed depending on the nematode-control level achieved. At selected time points, the mean number of trichostrongylid eggs, serum pepsinogen concentration and Ostertagia antibody levels were significantly higher in animals with poor performance. ROC analyses showed that anthelmintic treatment of animals with a Dwgt of <0.75 kg/day by mid-season had a sensitivity of approximately 70% and a specificity of approximately 50%. It thus seems feasible to base a targeted selective treatment for FSG cattle on Dwgt recorded approximately 4-8 weeks after turn-out, provided that it is accepted that some animals will be dewormed without indication. This now needs to be tested in controlled field trials.

Atmospheric deposition, retention, and stream export of dioxins and PCBs in a pristine boreal catchment

The mass-balance between diffuse atmospheric deposition of organic pollutants, amount of pollutants retained by the terrestrial environment, and levels of pollutants released to surface stream waters was studied in a pristine northern boreal catchment. This was done by comparing the input of atmospheric deposition of polychlorinated dibenzo-p-dioxins and furans (PCDD/Fs) and PCBs with the amounts exported to surface waters. Two types of deposition samplers were used, equipped with a glass fibre thimble and an Amberlite sampler respectively. The measured fluxes showed clear seasonality, with most of the input and export occurring during winter and spring flood, respectively. The mass balance calculations indicates that the boreal landscape is an effective sink for PCDD/Fs and PCBs, as 96.0-99.9 % of received bulk deposition was retained, suggesting that organic pollutants will continue to impact stream water in the region for an extended period of time.

Twelve-year interannual and seasonal variability of stream carbon export from a boreal peatland catchment

Understanding stream carbon export dynamics is needed to accurately predict how the carbon balance of peatland catchments will respond to climatic and environmental change. We used a 12year record ...

Performance of second-season grazing cattle following different levels of parasite control in their first grazing season.

A 3-year grazing trial was performed during 2003-2005 on a commercial steer-producing farm in Sweden to study performance of second-season grazing (SSG) cattle following different levels of parasite exposure during their first grazing season. Initially, groups of 10 first-season grazing (FSG) cattle were each year assigned to four parasite control strategies: (1) turn-out onto pasture that during the previous year was grazed by SSG cattle, followed by a mid-July move to aftermath, (2) supplementation with concentrate and roughage for 4 weeks after turn-out, (3) no treatment, or (4) anthelmintic treatment (injectable doramectin) every fourth week. All animals were set stocked, except for those in group one. Next spring and following housing the cattle were turned out for their second grazing season onto approximately 25 ha communal pasturelands as a common mob. Weighing, faecal sampling and blood collection were performed at turn-out and then every 4 weeks for the 20-week grazing season. Faecal samples were also collected on day 10 after turn-out for detection of coccidian oocysts. Antibodies to Dictyocaulus viviparus were analysed at the time of their second housing period, and when elevated levels were recorded, stored serum samples from seropositive animals were analysed retrospectively. Results showed early-season weight losses of up to 47 kg in the SSG cattle. However, faecal egg counts were generally low and there was no correlation between SSG performance and treatment history as FSG cattle. Still, cumulative egg counts were significantly higher in animals that had been treated with anthelmintic as FSG cattle but serum pepsinogen concentrations showed no significant differences and the output of Eimeria alabamensis oocysts seldom exceeded 10,000 oocysts per gram faeces. Antibodies to D. viviparus were observed from July 2004 and from June 2005 but not in 2003. In 2004 and 2005, 64% and 83% of the animals, respectively, were seropositive for D. viviparus. It is concluded that weight gain penalties resulting from different levels of parasite infections the first grazing season remained during the second grazing season in 2004 and 2005 but no differences in weight gain could be identified in SSG cattle that had experienced different parasite control measures during their first grazing season. This was under conditions where the level of pasture infectivity was low to moderate during the second grazing period. However, D. viviparus was demonstrated in SSG animals during the two last years of the study and was an important confounding factor.

Holocene carbon and nitrogen accumulation rates in a boreal oligotrophic fen

The contemporary role of mires in carbon exchange with the atmosphere is intensely debated. Thus, understanding the variation in Holocene peat accumulation is particularly important. We investigated carbon (C) and nitrogen (N) accumulation rates and their potential controls during the Holocene at the oligotrophic fen Degerö Stormyr (64°11′N, 19°33′E, 270 m a.s.l.), Sweden. The peat stratigraphy was dominated by remains of Eriophorum spp. and oligotrophic–mesotrophic Sphagnum spp. The long-term rate of C accumulation (LORCA) was 13.7 ± 5.5 (SD) g C m−2 yr−1, while the long-term rate of N accumulation (LORNA) was 0.28 ± 0.14 (SD) g N m−2 yr−1. Carbon and N accumulation rates exhibited similar variations that coincided with major changes in botanical composition. The botanical composition of the peat also had a major impact on the percentage of amorphous peat (a proxy for degree of decomposition). Bulk δ13C values increased with decreasing C content, which probably reflected the relative increase of 13C depleted compounds in the peat during decomposition. Different plant groups exhibited different relationships, likely due to different δ13C signatures of initial litters from Eriophorum spp. and Sphagnum spp. The δ15N values increased significantly with decreasing C:N ratio in bulk peat, likely reflecting preferential uptake of 14N by plants concomitant with nitrogen mineralization. Here, we demonstrate the importance of botanical composition in affecting C and N accumulation rates under a changing climate and suggest that primary production drives the variation in rates of accumulation. Furthermore, we point out the importance of including 13C and 15N signatures in the analysis of peat stratigraphies to advance interpretation of Holocene peat growth and decay.