1H NMR study of the role of heme carboxylate side chains in modulating heme pocket structure and the mechanism of reconstitution of cytochrome b5

1H nuclear magnetic resonance spectroscopy was used to assign the hyperfine-shifted resonances and determine the position of a side chain in the heme cavity of wild-type rat apocytochrome b5 reconstituted with a series of synthetic hemins possessing systematically perturbed carboxylate side chains. The hemins included protohemin derivatives with individually removed or pairwise shortened and lengthened carboxylate side chains, as well as (propionate)n(methyl)8-nporphine-iron(III) isomers with n = 1-3 designed to force occupation of nonnative propionate sites. The resonance assignments were effected on the basis of available empirical heme contact shift correlations and steady-state nuclear Overhauser effect measurements in the low-spin oxidized proteins. The failure to detect holoproteins with certain hemins dictates that the stable holoproteins, unlike the case of myoglobin, demand the axial iron-His bonds and cannot accommodate carboxylate side chains at interior positions in the binding pocket. Hence, the heme pocket interior in cytochrome b5 is judged much less polar and less sterically accommodating than that of myoglobin. The propionate occupational preference was greatest as the native 7-propionate site, but also possible at the nonnative crystallographic 5-methyl or 8-methyl positions. Only for a propionate at the crystallographic 8-methyl position was a significant perturbation of the native molecular/electronic structure observed, and this was attributed to an alternative propionate-protein hydrogen bond at the crystallographic 8-methyl position. The structures of the transient protein complexes detected only shortly after reconstitution reveal that the initial encounter complexes during assembly of holoprotein from apoprotein and hemin involve one of the two alternate propionate-protein links at either the 7-propionate or native 8-methyl position. In a monopropionate hemin, this leads to the characterization of a new type of heme orientational disorder involving rotation about a N-Fe-N axis.     

Caffeine Junkie: an Unprecedented Glutathione S-Transferase-Dependent Oxygenase Required for Caffeine Degradation by Pseudomonas putida CBB5

Caffeine and other N-methylated xanthines are natural products found in many foods, beverages, and pharmaceuticals. Therefore, it is not surprising that bacteria have evolved to live on caffeine as a sole carbon and nitrogen source. The caffeine degradation pathway of Pseudomonas putida CBB5 utilizes an unprecedented glutathione-S-transferase-dependent Rieske oxygenase for demethylation of 7-methylxanthine to xanthine, the final step in caffeine N-demethylation. The gene coding this function is unusual, in that the iron-sulfur and non-heme iron domains that compose the normally functional Rieske oxygenase (RO) are encoded by separate proteins. The non-heme iron domain is located in the monooxygenase, ndmC, while the Rieske [2Fe-2S] domain is fused to the RO reductase gene, ndmD. This fusion, however, does not interfere with the interaction of the reductase with N₁- and N₃-demethylase RO oxygenases, which are involved in the initial reactions of caffeine degradation. We demonstrate that the N₇-demethylation reaction absolutely requires a unique, tightly bound protein complex composed of NdmC, NdmD, and NdmE, a novel glutathione-S-transferase (GST). NdmE is proposed to function as a noncatalytic subunit that serves a structural role in the complexation of the oxygenase (NdmC) and Rieske domains (NdmD). Genome analyses found this gene organization of a split RO and GST gene cluster to occur more broadly, implying a larger function for RO-GST protein partners.     

Bio-control activity of Purpureocillium lilacinum strains in managing root-knot disease of tomato caused by Meloidogyne incognita

The potential of 24 indigenous isolates of Purpureocillium lilacinum (Paecilomyces lilacinus) (Thom) Samson collected from different agro-climatic zones of India was investigated against the root-knot nematode, Meloidogyne incognita. The studies were conducted in vitro (larvicidal, ovicidal and egg-parasitising capacity) and under naturally infested field conditions with selected strains. Repeated field trials were conducted with talc-based preparations of fungal strains at 10 kg ha^?1, which were applied mixed in farm yard manure (FYM) at 1.5 t ha^?1. Results (in vitro) showed that all tested isolates were capable to parasitise eggs, inhibit egg hatching and cause juvenile mortality of M. incognita at various levels. Based on the performance under in vitro studies, eight isolates (NDPL-01, ANDPL-02, SHGPL-03, HYBPL-04, AHDPL-05, PTNPL-06, SNGPL-07 and VNSPL-08) were re-tested to confirm the results. HYBDPL-04 was found causing highest mortality (80%), inhibition of egg hatching (90%) as well as parasitisation of M. incognita eggs (75%). Under field trials also, the best protection of root-knot disease of tomato (Lycopersicon esculentum L.), in terms of reduction of galls (61%) and reproductive factor (P_f/P_i (RF) = 0.2) was achieved through application of HYBDPL-04 + FYM compared to control and other tested isolates. It also enhanced marketable yield of tomato up to 43%. It is concluded that the HYBDPL-04 strain of P. lilacinum is highly effective for management of root-knot disease of tomato under naturally infested field conditions. It is the isolate which produced the maximum number of metabolites which were extracted through high pressure liquid chromatography.     

Economic and environmental impact assessments of a stand-alone napier grass-fired combined heat and power generation system in the southeastern US

The International Journal of Life Cycle Assessment - Napier grass, one of the high yield perennial energy crops can be grown on marginal lands with minimal inputs, but with increased soil carbon...     

Azadirachta indica and Ocimum sanctum leaf extracts alleviate arsenic toxicity by reducing arsenic uptake and improving antioxidant system in rice seedlings

In the present study the potentials of aqueous extracts of the two plants, neem (Azadirachta indica) and Tulsi (Ocimum sanctum) were examined in alleviating arsenic toxicity in rice (Oryza sativa L.) plants grown in hydroponics. Seedlings of rice grown for 8 days in nutrient solution containing 50 μM sodium arsenite showed decline in growth, reduced biomass, altered membrane permeability and increased production of superoxide anion (O2·−), H2O2 and hydroxyl radicals (·OH). Increased lipid peroxidation marked by elevated TBARS (thiobarbituric acid reactive substances) level, increased protein carbonylation, alterated levels of ascorbate, glutathione and increased activities of enzymes SOD (superoxide dismutase), CAT (catalase), APX (ascorbate peroxidase) and GPX (glutathione peroxidase) were noted in the seedlings on As treatment. Exogenously added leaf aqueous extracts of Azadirachta indica (0.75 mg mL−1, w/v) and Ocimum sanctum (0.87 mg mL−1, w/v) in the growth medium considerably alleviated As toxicity effects in the seedlings, marked by reduced As uptake, restoration of membrane integrity, reduced production of ROS, lowering oxidative damage and restoring the levels of ascorbate, glutathione and activity levels of antioxidative enzymes. Arsenic uptake in the seedlings declined by 72.5% in roots and 72.8% in shoots, when A. indica extract was present in the As treatment medium whereas with O. sanctum extract, the uptake declined by 67.2% in roots and 70.01% in shoots. Results suggest that both A. indica and O. sanctum aqueous extracts have potentials to alleviate arsenic toxicity in rice plants and that A. indica can serve as better As toxicity alleviator compared to O. sanctum.     

Correction to: Expression of the entomotoxic Cocculus hirsutus trypsin inhibitor (ChTI) gene in transgenic chickpea enhances its underlying resistance against the infestation of Helicoverpa armigera and Spodoptera litura

Plant Cell, Tissue and Organ Culture (PCTOC) - A correction to this paper has been published: https://doi.org/10.1007/s11240-021-02054-x