Topoisomerase I (TOP1) dynamics: conformational transition from open to closed states

Eukaryotic topoisomerases I (TOP1) are ubiquitous enzymes removing DNA torsional stress. However, there is little data concerning the three-dimensional structure of TOP1 in the absence of DNA, nor how the DNA molecule can enter/exit its closed conformation. Here, we solved the structure of thermostable archaeal Caldiarchaeum subterraneum CsTOP1 in an apo-form. The enzyme displays an open conformation resulting from one substantial rotation between the capping (CAP) and the catalytic (CAT) modules. The junction between these two modules is a five-residue loop, the hinge, whose flexibility permits the opening/closing of the enzyme and the entry of DNA. We identified a highly conserved tyrosine near the hinge as mediating the transition from the open to closed conformation upon DNA binding. Directed mutagenesis confirmed the importance of the hinge flexibility, and linked the enzyme dynamics with sensitivity to camptothecin, a TOP1 inhibitor targeting the TOP1 enzyme catalytic site in the closed conformation.

Evaluation of Compost and Biochar to Mitigate Chlorpyrifos Pollution in Soil and Their Effect on Soil Enzyme Dynamics

The widespread environmental contamination of chlorpyrifos (CP) has raised human health concerns and necessitated cost-effective methods for its remediation. The current study evaluated the degradation behavior of CP in compost and biochar amended and unamended (original and sterilized) soils in an incubation trial. Two levels of CP (100 and 200 mg kg−1), compost and biochar (0.50%) were applied, and soil was collected at different time intervals. At the higher CP level (200 mg kg−1), CP a showed lower degradation rate (ƙ = 0.0102 mg kg−1 d−1) compared with a low CP level (ƙ = 0.0173 mg kg−1 d−1). The half-lives of CP were 40 and 68 days for CP at 100 and 200 mg kg−1 in original soil, respectively, and increased to 94 and 141 days in sterilized soils. CP degradation was accelerated in compost amended soils, while suppressed in biochar amended soils. Lower half lives of 20 and 37 days were observed with compost application at CP 100 and 200 mg kg−1 doses, respectively. The activities of soil enzymes were considerably affected by the CP contamination and significantly recovered in compost and biochar amended soils. In conclusion, the application of organic amendments especially compost is an important strategy for the remediation of CP contaminated soil.

X-ray free-electron laser studies reveal correlated motion during isopenicillin N synthase catalysis

Isopenicillin N synthase (IPNS) catalyzes the unique reaction of l-δ-(α-aminoadipoyl)-l-cysteinyl-d-valine (ACV) with dioxygen giving isopenicillin N (IPN), the precursor of all natural penicillins and cephalosporins. X-ray free-electron laser studies including time-resolved crystallography and emission spectroscopy reveal how reaction of IPNS:Fe(II):ACV with dioxygen to yield an Fe(III) superoxide causes differences in active site volume and unexpected conformational changes that propagate to structurally remote regions. Combined with solution studies, the results reveal the importance of protein dynamics in regulating intermediate conformations during conversion of ACV to IPN. The results have implications for catalysis by multiple IPNS-related oxygenases, including those involved in the human hypoxic response, and highlight the power of serial femtosecond crystallography to provide insight into long-range enzyme dynamics during reactions presently impossible for nonprotein catalysts.

Does It Pay Off to Explicitly Link Functional Gene Expression to Denitrification Rates in Reaction Models?

Environmental omics and molecular-biological data have been proposed to yield improved quantitative predictions of biogeochemical processes. The abundances of functional genes and transcripts relate to the number of cells and activity of microorganisms. However, whether molecular-biological data can be quantitatively linked to reaction rates remains an open question. We present an enzyme-based denitrification model that simulates concentrations of transcription factors, functional-gene transcripts, enzymes, and solutes. We calibrated the model using experimental data from a well-controlled batch experiment with the denitrifier Paracoccous denitrificans. The model accurately predicts denitrification rates and measured transcript dynamics. The relationship between simulated transcript concentrations and reaction rates exhibits strong non-linearity and hysteresis related to the faster dynamics of gene transcription and substrate consumption, relative to enzyme production and decay. Hence, assuming a unique relationship between transcript-to-gene ratios and reaction rates, as frequently suggested, may be an erroneous simplification. Comparing model results of our enzyme-based model to those of a classical Monod-type model reveals that both formulations perform equally well with respect to nitrogen species, indicating only a low benefit of integrating molecular-biological data for estimating denitrification rates. Nonetheless, the enzyme-based model is a valuable tool to improve our mechanistic understanding of the relationship between biomolecular quantities and reaction rates. Furthermore, our results highlight that both enzyme kinetics (i.e., substrate limitation and inhibition) and gene expression or enzyme dynamics are important controls on denitrification rates.

Structural and biochemical analysis of ATPase activity and EsxAB substrate binding of M. tuberculosis EccCb1 enzyme

The EccC enzyme of M. tuberculosis ESX-1 system is a promising target for antivirulence drug development. The EccC enzyme comprises two polypeptides (i) EccCa1, a membrane bound enzyme having two ATPase domains D2 & D3 (ii) cytosolic EccCb, which contains two ATPase domains. In current study, we have analyzed the low-resolution structure of EccCb1, performed ATPase activity and EsxAB substrate binding analysis. The EccCb1 enzyme eluted as oligomer from size exclusion column and small angle X-ray scattering analysis revealed the double hexameric structure in solution. The EccCb1 enzyme showed catalytic efficiency (kcat/KM)~ 0.020 micromolar-1 min-1, however ~3.7 fold lower than its D2 and ~1.7 fold lower than D3 domains respectively. The D2 and D3 domains exhibited the ATPase activity and mutation of residues involved in ATP+Mg2+ binding have yielded 56-94% reduction in catalytic efficiency for both D2 and D3 domains. The EccCb1 binds the EsxAB substrate with KD ~ 11.4 nM via specific groove located at C-terminal region of D3 domain. ATP binding to EccCb1 enhanced the EsxAB substrate binding by ~ 3 fold, indicating ATPase energy involvement in EsxAB substrate translocation. We modeled the dodecameric EccCb1+EsxAB+ ATP+Mg2+ complex, which showed the binding pockets involved in ATP+Mg2+ and EsxAB substrate binding. The enzyme dynamics involved in ATP+Mg2+ and EsxAB substrate recognition were identified and showed the enhanced stability of EccCb1 enzyme as a result of ligand binding. Overall, our structural and biochemical analysis showed the low-resolution structure and mechanism involved in ATPase activity and EsxAB substrate binding and dynamics involved in EsxAB substrate and ATP+Mg2+ recognition. Overall, our structural and biochemical data on EccCb1 will contribute significantly in development of antivirulence inhibitors, which will prevent virulence factor secretion by M. tuberculosis ESX-1 system.

A stochastic model of hippocampal synaptic plasticity with geometrical readout of enzyme dynamics

Synaptic plasticity rules used in current computational models of learning are generally insensitive to physiological factors such as spine voltage, animal age, extracellular fluid composition, and body temperature, limiting their predictive power. Here, we built a biophysically detailed synapse model inclusive of electrical dynamics, calcium-dependent signaling via CaMKII and Calcineurin (CaN) activities. The model combined multi-timescale variables, milliseconds to minutes, and intrinsic noise from stochastic ion channel gating. Analysis of the trajectories of joint CaMKII and CaN activities yielded an interpretable geometrical readout that fitted the synaptic plasticity outcomes of nine published ex vivo experiments covering various spike-timing and frequency-dependent plasticity induction protocols, animal ages, and experimental conditions. Using this new approach, we then generated maps predicting plasticity outcomes across the space of these stimulation conditions. Finally, we tested the model's robustness to in vivo-like spike time irregularity, showing that it significantly alters plasticity outcomes.

Chemical Properties, Microbial Biomass and Enzyme Dynamics of Post-Harvest Soil on Transplanted Rice with Effect of Organic Amendments

A field experiment was conducted at the farm of Tamil Nadu Agricultural University, Coimbatore during kharif season to study the effect of soil chemical properties, microbial biomass and soil enzyme dynamics on transplanted rice with organic amendments. Rice CO(R) 48 was used as a test variety. The experiment was laid out in randomized block design (RBD) with three replications and nine treatments. The treatment consists Recommended Dose of Fertilizers 100% NPK (150 : 50 : 50 kg ha-1) through inorganic fertilizers, Based on N equivalent basis, required quantities of organic manures (Dhaincha, vermicompost and Farmyard Manure @ 50% and 100%) were incorporated into the soil one week before transplanting of rice. The P and K requirement was supplied separately through inorganic sources as per treatment schedule. In the present investigation, an attempt was made to examine the influence of different levels and sources of fertilization on dynamics of soil chemical properties, microbial biomass and enzyme activities under anaerobic rice cultivation. Soil samples were taken before the start of experiment and harvest of rice for analysis of soil chemical characteristics, microbial biomass and enzyme activity. The results revealed that maximum microbial population (bacteria, fungal & actinomycetes), soil enzyme dynamics (urease, dehydrogenase & soil phosphatase activity), soil available macro nutrients (N, P, K) & micro nutrients (Fe, Zn, Mn, Cu) were significantly influenced with application of 100% N through dhaincha + balance P & K through inorganic fertilizers followed by application of 100% NPK through inorganic fertilizers + 6.25 t dhaincha. Least enzyme activity, microbial population & soil available nutrients of rice were registered in absolute control.