A pair of -cyano analogues of decarboxylated S-adenosylmethionine (2a and 2b) were synthesized as potential enzyme activated, irreversible inhibitors of the[pyruvoyl enzyme S-adenosylmethionine decarboxylase (AdoMet-DC). Each of these analogues acts as an irreversible inactivator for ADoMet-DC from Escherichia coli (IC50 values of 9 and 50 μM, respectively). These analogues also inactivate human AdoMet-DC, with KI values of 246.6 and 7.2 μM, and kinact values of 0.29 and 0.03 min−1, respectively. 相似文献
The conformationally restricted S-adenosylmethionine analogue AdoMac (S-(5′-deoxy-5′-adenosyl)-1-ammonio-4-methylsulfonio-2-cyclopentene has been shown to act as an enzyme activated, irreversible inhibitor of theEscherichia coli form of the enzyme S-adenosylmethionine decarboxylase. Inactivation of the enzyme is presumably initiated by formation of an imine linkage between the inhibitor and the terminal pyruvate of the enzyme, followed by base-catalyzed elimination of methylthioadenosine and generation of a latent electrophile. Removal of the driving force for the elimination of methylthioadenosine resulted in a reversibly binding inhibitor. Thus, the thioether analogue corresponding to AdoMac, and the corresponding dihydro derivative (H2-AdoMac), reversibly inhibit the enzyme. AdoMac was resolved into its four pure diastereomeric forms, and each diastereomer was evaluated as an irreversible inhibitor of the enzyme. The KI values for the individual diastereomers range between 3.83 and 39.6 μM, with the cis-1S,4R diastereomer being the most potent inhibitor. However, the kinact values for the four diastereomers are not significantly different, suggesting that the binding of each diastereomer to the enzyme is configuration-dependent, while the subsequent inactivation likely proceeds through a single intermediate which is formed from each of the four diastereomers. Since each pure diastereomer represents a distinct conformational mimic exhibiting restricted sidechain rotation, the data suggests that these and related analogues may be useful as conformational probes for the catalytic site of AdoMet-DC. 相似文献
This study examined the effects of nitrogen (N) fertilizer reduction on the carbon (C) metabolism and yield of Coreopsis tinctoria. A two-year (2020–2021) hydroponic experiment was conducted in accordance with a randomized complete group design with five N levels [0.875 mM Ca(NO3)2 (N1), 1.750 mM Ca(NO3)2 (N2), 3.500 mM Ca(NO3)2 (N3), 7.000 mM Ca(NO3)2 (N4), and 14.000 mM Ca(NO3)2 (N5)] and three replications. The results showed that low N significantly affected the functional leaf weight, C metabolism, and flower bud (or flower) numbers of C. tinctoria at harvest. Lower-N levels, especially those of the N2 treatment, significantly increased Rubisco, sucrose synthase (SS), sucrose phosphate synthase (SPS), soluble acid invertase (SAI), glucose 6-phosphate dehydrogenase (G6PDH), and 6-phosphogluconate dehydrogenase (6PGDH) activity and maintained the flower number of C. tinctoria. In addition, the balance of carbohydrates (sucrose, starch, glucose, and fructose) and ATP contents was more efficiently maintained under relatively low-N levels. These findings might suggest that reduced application of N fertilizer affects the C metabolism of leaves and maintains the number of flowers in Coreopsis tinctoria. Applying relatively low-N fertilizer levels is also a promising cultivation strategy for C. tinctoria.
Biochar and manure can be used for sustainable land management. However, little is known about how soil amendments might affect surface and belowground microbial processes and subsequent wood decomposition. In a split-split-split plot design, we amended soil with two rates of manure (whole plot; 0 and 9 Mg ha−1) and biochar (split plot; 0 and 10 Mg ha−1). Wood stakes of three species (hybrid poplar, triploid Populus tomentosa Carr.; aspen, Populus tremuloides Michx.; and pine, Pinus taeda L.) were placed in two positions (horizontally on the soil surface, and inserted vertically in the mineral soil), which served as a substrate for fungal growth. In 3 years, the decomposition rate (density loss), moisture content, and fungal community (via high-throughput sequencing methods) of stakes were evaluated. Results indicated that biochar and/or manure increased the wood stake decomposition rates, moisture content, and operational taxonomic unit abundance. However, the richness and diversity of fungi were dependent on wood stake position (surface > mineral), species (pine > the two Populus), and sample dates. This study highlights that soil amendment with biochar and/or manure can alter the fungal community, which in turn can enhance an important soil process (i.e., decomposition). 相似文献
R-1,3-butanediol (R-1,3-BDO) is an important chiral intermediate of penem and carbapenem synthesis. Among the different synthesis methods to obtain pure enantiomer R-1,3-BDO, oxidation–reduction cascades catalysed by enzymes are promising strategies for its production. Dehydrogenases have been used for the reduction step, but the enantio-selectivity is not high enough for further organic synthesis efforts. Here, a short-chain carbonyl reductase (LnRCR) was evaluated for the reduction step and developed via protein engineering. After docking result analysis with the substrate 4-hydroxy-2-butanone (4H2B), residues were selected for virtual mutagenesis, their substrate-binding energies were compared, and four sites were selected for saturation mutagenesis. High-throughput screening helped identify a Ser154Lys mutant which increased the catalytic efficiency by 115% compared to the parent enzyme. Computer-aided simulations indicated that after single residue replacement, movements in two flexible areas (VTDPAF and SVGFANK) facilitated the volumetric compression of the 4H2B-binding pocket. The number of hydrogen bonds between the stabilized 4H2B-binding pocket of the mutant enzyme and substrate was higher (from four to six) than the wild-type enzyme, while the substrate-binding energy was decreased (from −17.0 kJ/mol to −29.1 kJ/mol). Consequently, the catalytic efficiency increased by approximately 115% and enantio-selectivity increased from 95% to 99%. Our findings indicate that compact and stable substrate-binding pockets are critical for enzyme catalysis. Lastly, the utilization of a microbe expressing the Ser154Lys mutant enzyme was proven to be a robust process to conduct the oxidation–reduction cascade at larger scales. 相似文献
Alterations in the amount of fibronectin and in the number of its receptors during myoblast differentiation of chicken embryo were investigated. The amount of fibronectin in the cell surface pool as measured by immunoblotting decreased during myogenesis To identify and characterize the fibronectin receptors on the myoblasts, the interactions of the 28,000 dalton (28 kDa) amino terminal fragment and 85,000 dalton (85 kDa) cell-binding fragment of fibronectin with my-oblasts were examined. The binding of the 28 kDa fragment was found to be time-dependent and reached a maximum level within 60 min. The unlabeled 28 kDa fragment inhibited the binding of the radioiodinated 28 kDa fragment, whereas the unlabeled 85 kDa fragment and antibody to integrin did not inhibit it, suggesting that the 28 kDa fragment interacts with the matrix assembly receptors but not with the cell adhesion receptors. There was a single class of 3.4 × 105 binding sites per cell with an apparent dissociation constant of 1.4 × 10?7 M on 30 hr old myoblasts. The specific binding of the radioiodinated 28 kDa fragment to myoblasts decreased as the fusion proceeded. This decrease of binding was consistent with the decrease in the amount of fibronectin. Furthermore, the levels of fibronectin and binding of the radioiodinated 28 kDa fragment in the fusion-blocked myoblasts by EGTA treatment appeared to remain constant. These results suggest that the decrease and/or loss of fibronectin during myoblast fusion is closely correlated with the alteration of fibronectin receptors and with the fusion of myoblasts. 相似文献
Complete mRNA sequence of transferrin from Galleria mellonella was obtained, and compared with those of other species. Until now, two types of insect transferrin were reported. Transferrins in cockroach and termite have two iron binding sites while those in most other insect groups, studied for the protein, have only one. It was suggested that the presence of two types of transferrin was related with transferrin evolution, because vertebrate transferrins have two iron binding sites, called N and C terminal lobe. It was shown that G. mellonella transferrin also has only one iron binding site (N terminal lobe), and the deduced amino acid sequence was most similar to those of Manduca sexta and Bombyx mori.相似文献
Summary Double fluorescent labeling, with fluorescein isothiocyanate (FITC)-labeled F(ab)2 specific for the heavy chain and R-phycoerythrin (R-PE)-labeled F(ab)2 specific for the light chain, was demonstrated as a convenient means for the accurate evaluation of a heterogeneous non-antibody-producing population. Furthermore, it could be used for monitoring the changes in each immunoglobulin (Ig) chain content of the cells during the batch culture, which will facilitate the study on antibody synthesis, assembly and secretion. 相似文献