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631 Publications visible to you, out of a total of 631
Kinase activity of ArcB from Escherichia coli is subject to regulation by both ubiquinone and demethylmenaquinone

Abstract (Expand)

Expression of the catabolic network in Escherichia coli is predominantly regulated, via oxygen availability, by the two-component system ArcBA. It has been shown that the kinase activity of ArcB is … controlled by the redox state of two critical pairs of cysteines in dimers of the ArcB sensory kinase. Among the cellular components that control the redox state of these cysteines of ArcB are the quinones from the cytoplasmic membrane of the cell, which function in 'respiratory' electron transfer. This study is an effort to understand how the redox state of the quinone pool(s) is sensed by the cell via the ArcB kinase. We report the relationship between growth, quinone content, ubiquinone redox state, the level of ArcA phosphorylation, and the level of ArcA-dependent gene expression, in a number of mutants of E. coli with specific alterations in their set of quinones, under a range of physiological conditions. Our results provide experimental evidence for a previously formulated hypothesis that not only ubiquinone, but also demethylmenaquinone, can inactivate kinase activity of ArcB. Also, in a mutant strain that only contains demethylmenaquinone, the extent of ArcA phosphorylation can be modulated by the oxygen supply rate, which shows that demethylmenaquinone can also inactivate ArcB in its oxidized form. Furthermore, in batch cultures of a strain that contains ubiquinone as its only quinone species, we observed that the ArcA phosphorylation level closely followed the redox state of the ubiquinone/ubiquinol pool, much more strictly than it does in the wild type strain. Therefore, at low rates of oxygen supply in the wild type strain, the activity of ArcB may be inhibited by demethylmenaquinone, in spite of the fact that the ubiquinones are present in the ubiquinol form.

Authors: P. Sharma, S. Stagge, M. Bekker, K. Bettenbrock, K. J. Hellingwerf

Date Published: 7th Oct 2013

Publication Type: Not specified

Kinetic characterization of arginine deiminase and carbamate kinase from Streptococcus pyogenes M49

Abstract (Expand)

Streptococcus pyogenes (group A Streptococcus, GAS) is an important human pathogen causing mild superficial infections of skin and mucous membranes, but also life-threatening systemic diseases. S. … pyogenes and other prokaryotic organisms use the arginine deiminase system (ADS) for survival in acidic environments. In this study, the arginine deiminase (AD), and carbamate kinase (CK) from S. pyogenes M49 strain 591 were heterologously expressed in E. coli DH5α, purified, and kinetically characterized. AD and CK from S. pyogenes M49 share high amino acid sequence similarity with the respective enzymes from Lactococcus lactis subsp. lactis IL1403 (45.6% and 53.5% identical amino acids) and Enterococcus faecalis V583 (66.8% and 66.8% identical amino acids). We found that the arginine deiminase of S. pyogenes is not allosterically regulated by the intermediates and products of the arginine degradation (E. g, ATP, citrulline, carbamoyl phosphate). The Km and Vmax values for arginine were 1.13±0.12 mM (mean ± SD) and 1.51±0.07 μmol/min/mg protein. The carbamate kinase is inhibited by ATP but unaffected by arginine and citrulline. The Km and Vmax values for ADP were 0.72±0.08 mM and 1.10±0.10 μmol/min/mg protein and the Km for carbamoyl phosphate was 0.65±0.07 mM. The optimum pH and temperature for both enzymes were 6.5 and 37°C, respectively.

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Date Published: 1st Jul 2013

Publication Type: Not specified

Kinetic characterization of two neuraminic acid synthases and evaluation of their application potential

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Neuraminic acid synthases are an important yet underexplored group of enzymes. Thus, in this research, we performed a detailed kinetic and stability analysis and a comparison of previously known … neuraminic acid synthase from Neisseria meningitidis, and a novel enzyme, PNH5, obtained from a metagenomic library. A systematic analysis revealed a high level of similarity of PNH5 to other known neuraminic acid synthases, except for its pH optimum, which was found to be at 5.5 for the novel enzyme. This is the first reported enzyme from this family that prefers an acidic pH value. The effect of different metal cofactors on enzyme activity, i.e. Co2+, Mn2+ and Mg2+, was studied systematically. The kinetics of neuraminic acid synthesis was completely elucidated, and an appropriate kinetic model was proposed. Enzyme stability study revealed that the purified enzyme exhibits changes in its structure during time as observed by differential light scattering, which cause a drop in its activity and protein concentration. The operational enzyme stability for the neuraminic acid synthase from N. meningitidis is excellent, where no activity drop was observed during the batch reactor experiments. In the case of PNH5, some activity drop was observed at higher concentration of substrates. The obtained results present a solid platform for the future application of these enzymes in the synthesis of sialic acids.

Authors: Mehmet Mervan Çakar, Nevena Milčić, Theofania Andreadaki, Simon Charnock, Wolf-Dieter Fessner, Zvjezdana Findrik Blažević

Date Published: 21st Aug 2024

Publication Type: Journal

Kinetic modeling identifies targets for engineering improved photosynthetic efficiency in potato (Solanum tuberosum cv. Solara).

Abstract (Expand)

Potato (Solanum tuberosum) is a significant non-grain food crop in terms of global production. However, its yield potential might be raised by identifying means to release bottlenecks within photosynthetic … metabolism, from the capture of solar energy to the synthesis of carbohydrates. Recently, engineered increases in photosynthetic rates in other crops have been directly related to increased yield - how might such increases be achieved in potato? To answer this question, we derived the photosynthetic parameters V(cmax) and J(max) to calibrate a kinetic model of leaf metabolism (e-Photosynthesis) for potato. This model was then used to simulate the impact of manipulating the expression of genes and their protein products on carbon assimilation rates in silico through optimizing resource investment among 23 photosynthetic enzymes, predicting increases in photosynthetic CO(2) uptake of up to 67%. However, this number of manipulations would not be practical with current technologies. Given a limited practical number of manipulations, the optimization indicated that an increase in amounts of three enzymes - Rubisco, FBP aldolase, and SBPase - would increase net assimilation. Increasing these alone to the levels predicted necessary for optimization increased photosynthetic rate by 28% in potato.

Authors: S. Vijayakumar, Y. Wang, G. Lehretz, S. Taylor, E. Carmo-Silva, S. Long

Date Published: 30th Jan 2024

Publication Type: Journal

Know when your numbers are significant

Abstract (Expand)

Experimental biologists, their reviewers and their publishers must grasp basic statistics, urges David L. Vaux, or sloppy science will continue to grow. "And, once in the lab, people generally …y just do what everyone else does, without always understanding why." (D. Vaux)

Author: David L. Vaux

Date Published: 1st Dec 2012

Publication Type: Journal

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