scholarly journals Sulfate adenylyl transferase kinetics and mechanisms of metabolic inhibitors of microbial sulfate respiration

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Hans K. Carlson ◽  
Matthew D. Youngblut ◽  
Steven A. Redford ◽  
Adam J. Williamson ◽  
John D. Coates
Keyword(s):  
Industrial Applications ◽  
Metabolic Inhibitors ◽  
Current Evidence ◽  
Ecological Studies ◽  
Adaptive Laboratory Evolution ◽  
Inhibitory Potency ◽  
Sulfate Reducing ◽  
Important Target ◽  
Mechanism Of Inhibition ◽  
Competitive Inhibitors

AbstractSulfate analog oxyanions that function as selective metabolic inhibitors of dissimilatory sulfate reducing microorganisms (SRM) are widely used in ecological studies and industrial applications. As such, it is important to understand the mode of action and mechanisms of tolerance or adaptation to these compounds. Different oxyanions vary widely in their inhibitory potency and mechanism of inhibition, but current evidence suggests that the sulfate adenylyl transferase/ATP sulfurylase (Sat) enzyme is an important target. We heterologously expressed and purified the Sat from the model SRM, Desulfovibrio alaskensis G20. With this enzyme we determined the turnover kinetics (kcat, KM) for alternative substrates (molybdate, selenate, arsenate, monofluorophosphate, and chromate) and inhibition constants (KI) for competitive inhibitors (perchlorate, chlorate, and nitrate). These measurements enable the first quantitative comparisons of these compounds as substrates or inhibitors of a purified Sat from a respiratory sulfate reducer. We compare predicted half-maximal inhibitory concentrations (IC50) based on Sat kinetics with measured IC50 values against D. alaskensis G20 growth and discuss our results in light of known mechanisms of sensitivity or resistance to oxyanions. This analysis helps with the interpretation of recent adaptive laboratory evolution studies and illustrates the value of interpreting gene–microbe–environment interactions through the lens of enzyme kinetics.

scholarly journals Sulfate adenylyl transferase kinetics and mechanisms of metabolic inhibitors of microbial sulfate respiration

2021 ◽  
Author(s):  
Hans K Carlson ◽  
Matthew D. Youngblut ◽  
Steven A Redford ◽  
Adam J Williamson ◽  
John Coates
Keyword(s):  
Industrial Applications ◽  
Metabolic Inhibitors ◽  
Current Evidence ◽  
Adaptive Laboratory Evolution ◽  
Inhibitory Potency ◽  
Sulfate Reducing ◽  
Important Target ◽  
Mechanism Of Inhibition ◽  
Competitive Inhibitors ◽  
Ic50 Values

Sulfate analog oxyanions that function as selective metabolic inhibitors of dissimilatory sulfate reducing microorganisms (SRM) are widely used in ecological studies and industrial applications. As such, it is important to understand the mode of action and mechanisms of tolerance or adaptation to these compounds. Different oxyanions vary widely in their inhibitory potency and mechanism of inhibition, but current evidence suggests that the sulfate adenylyl transferase/ATP sulfurylase (Sat) enzyme is an important target. We heterologously expressed and purified the Sat from the model SRM, Desulfovibrio alaskensis G20. With this enzyme we determined the turnover kinetics (kcat, KM) for alternative substrates (molybdate, selenate, arsenate, monofluorophosphate, and chromate) and inhibition constants (KI) for competitive inhibitors (perchlorate, chlorate, and nitrate). These measurements enable the first quantitative comparisons of these compounds as substrates or inhibitors of a purified Sat from a respiratory sulfate reducer. We compare predicted half-maximal inhibitory concentrations (IC50) based on Sat kinetics with measured IC50 values against D. alaskensis G20 growth and discuss our results in light of known mechanisms of sensitivity or resistance to oxyanions. This analysis helps with the interpretation of recent adaptive laboratory evolution studies and illustrates the value of interpreting gene-microbe-environment interactions through the lens of enzyme kinetics.

scholarly journals In silico-guided engineering of Pseudomonas putida towards growth under micro-oxic conditions

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Linde F. C. Kampers ◽  
Ruben G. A. van Heck ◽  
Stefano Donati ◽  
Edoardo Saccenti ◽  
Rita J. M. Volkers ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
In Silico ◽  
Redox Balance ◽  
Industrial Applications ◽  
Strictly Aerobic ◽  
Class Iii ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
Atp Production ◽  
Lactobacillus Lactis

Abstract Background Pseudomonas putida is a metabolically versatile, genetically accessible, and stress-robust species with outstanding potential to be used as a workhorse for industrial applications. While industry recognises the importance of robustness under micro-oxic conditions for a stable production process, the obligate aerobic nature of P. putida, attributed to its inability to produce sufficient ATP and maintain its redox balance without molecular oxygen, severely limits its use for biotechnology applications. Results Here, a combination of genome-scale metabolic modelling and comparative genomics is used to pinpoint essential $$\text {O}_{2}$$ O 2 -dependent processes. These explain the inability of the strain to grow under anoxic conditions: a deficient ATP generation and an inability to synthesize essential metabolites. Based on this, several P. putida recombinant strains were constructed harbouring acetate kinase from Escherichia coli for ATP production, and a class I dihydroorotate dehydrogenase and a class III anaerobic ribonucleotide triphosphate reductase from Lactobacillus lactis for the synthesis of essential metabolites. Initial computational designs were fine-tuned by means of adaptive laboratory evolution. Conclusions We demonstrated the value of combining in silico approaches, experimental validation and adaptive laboratory evolution for microbial design by making the strictly aerobic Pseudomonas putida able to grow under micro-oxic conditions.

scholarly journals Targeting cancer stem cells from a metabolic perspective

2020 ◽  
Vol 245 (5) ◽  
pp. 465-476 ◽  
Author(s):  
Yao-An Shen ◽  
Siao-Cian Pan ◽  
I Chu ◽  
Ruo-Yun Lai ◽  
Yau-Huei Wei
Keyword(s):  
Stem Cells ◽  
Cancer Stem Cells ◽  
Defense Mechanism ◽  
Therapeutic Targets ◽  
Metabolic Inhibitors ◽  
Current Evidence ◽  
Special Focus ◽  
Self Renewal ◽  
Atp Production ◽  
Anticancer Treatment

The process of cancer development and progression is driven by distinct subsets of cancer stem cells (CSCs) that contribute the self-renewal capacity as the major impetus to the metastatic dissemination and main impediments in cancer treatment. Given that CSCs are so scarce in the tumor mass, there are debatable points on the metabolic signatures of CSCs. As opposed to differentiated tumor progenies, CSCs display exquisite patterns of metabolism that, depending on the type of cancer, predominately rely on glycolysis, oxidative metabolism of glutamine, fatty acids, or amino acids for ATP production. Metabolic heterogeneity of CSCs, which attributes to differences in type and microenvironment of tumors, confers CSCs to have the plasticity to cope with the endogenous mitochondrial stress and exogenous microenvironment. In essence, CSCs and normal stem cells are like mirror images of each other in terms of metabolism. To achieve reprogramming, CSCs not only need to upregulate their metabolic engine for self-renewal and defense mechanism, but also expedite the antioxidant defense to sustain the redox homeostasis. In the context of these pathways, this review portrays the connection between the metabolic features of CSCs and cancer stemness. Identification of the metabolic features in conferring resistance to anticancer treatment dictated by CSCs can enhance the opportunity to open up a new therapeutic dimension, which might not only improve the effectiveness of cancer therapies but also annihilate the whole tumor without recurrence. Henceforth, we highlight current findings of potential therapeutic targets for the design of alternative strategies to compromise the growth, drug resistance, and metastasis of CSCs by altering their metabolic phenotypes. Perturbing the versatile skills of CSCs by barricading metabolic signaling might bring about plentiful approaches to discover novel therapeutic targets for clinical application in cancer treatments. Impact statement This minireview highlights the current evidence on the mechanisms of pivotal metabolic pathways that attribute to cancer stem cells (CSCs) with a special focus on developing metabolic strategies of anticancer treatment that can be exploited in preclinical and clinical settings. Specific metabolic inhibitors that can overwhelm the properties of CSCs may impede tumor recurrence and metastasis, and potentially achieve a permanent cure of cancer patients.

Food additive emulsifiers and their impact on gut microbiome, permeability and inflammation: mechanistic insights in inflammatory bowel disease

2020 ◽  
Author(s):  
Aaron S Bancil ◽  
Alicia M Sandall ◽  
Megan Rossi ◽  
Benoit Chassaing ◽  
James O Lindsay ◽  
...  
Keyword(s):  
Inflammatory Bowel Disease ◽  
Bowel Disease ◽  
Animal Studies ◽  
Intervention Studies ◽  
Dietary Intervention ◽  
Food Additive ◽  
Current Evidence ◽  
Global Burden ◽  
Ecological Studies ◽  
Inflammatory Bowel

Abstract The global burden of inflammatory bowel disease (IBD) has increased over the 21 st century. Despite multiple studies investigating the pathogenesis of IBD, the causative mechanisms pertaining to the increased prevalence remain unclear. There is growing evidence that aspects of a ‘Western diet’ increase the risk of developing IBD. More recently, evidence implicating dietary emulsifiers has accumulated, with ecological studies showing a positive correlation with inflammatory bowel disease and emulsifier consumption. Further to these, cell and animal studies have demonstrated plausible mechanisms by which dietary emulsifiers may contribute to IBD pathogenesis through mechanisms including: promotion of pro-inflammatory intestinal microbiota; disruption of mucus architecture; increased intestinal permeability; activation of inflammatory pathways and disruption of the cell cycle. This review critically analyses the current evidence for these mechanisms that may be of pathological relevance to IBD, evaluates recent dietary trials, acknowledges the challenges of dietary intervention studies and gives an overview of ongoing and future clinical trials in this important area.

scholarly journals Identification of broad-based HIV-1 protease inhibitors from combinatorial libraries

2010 ◽  
Vol 429 (3) ◽  
pp. 527-532 ◽  
Author(s):  
Max W. Chang ◽  
Michael J. Giffin ◽  
Rolf Muller ◽  
Jeremiah Savage ◽  
Ying C. Lin ◽  
...  
Keyword(s):  
Combinatorial Libraries ◽  
Wild Type ◽  
Virion Assembly ◽  
Chemical Libraries ◽  
Mechanism Of Inhibition ◽  
Competitive Mechanism ◽  
Starting Point ◽  
Competitive Inhibitors ◽  
Protein Nucleic Acid ◽  
Hiv 1

Clinically approved inhibitors of the HIV-1 protease function via a competitive mechanism. A particular vulnerability of competitive inhibitors is their sensitivity to increases in substrate concentration, as may occur during virion assembly, budding and processing into a mature infectious viral particle. Advances in chemical synthesis have led to the development of new high-diversity chemical libraries using rapid in-solution syntheses. These libraries have been shown previously to be effective at disrupting protein–protein and protein–nucleic acid interfaces. We have screened 44000 compounds from such a library to identify inhibitors of the HIV-1 protease. One compound was identified that inhibits wild-type protease, as well as a drug-resistant protease with six mutations. Moreover, analysis of this compound suggests an allosteric non-competitive mechanism of inhibition and may represent a starting point for an additional strategy for anti-retroviral therapy.

scholarly journals Synergistic Inhibition of Microbial Sulfide Production by Combinations of the Metabolic Inhibitor Nitrite and Biocides

2006 ◽  
Vol 72 (12) ◽  
pp. 7897-7901 ◽  
Author(s):  
E. Anne Greene ◽  
Veronique Brunelle ◽  
Gary E. Jenneman ◽  
Gerrit Voordouw
Keyword(s):  
Inhibitory Concentration ◽  
Cost Effective ◽  
Metabolic Inhibitors ◽  
Effective Control ◽  
Metabolic Inhibitor ◽  
Specific Inhibition ◽  
Synergistic Inhibition ◽  
Sulfate Reducing ◽  
Dissimilatory Sulfite Reductase ◽  
Reducing Bacteria

ABSTRACT MICs of six broad-spectrum biocides and two specific metabolic inhibitors and fractional inhibitory concentration indexes (FICIs) for controlling a sulfide-producing consortium were determined. Nitrite was synergistic (FICI < 1) with all but one biocide due to its specific inhibition of dissimilatory sulfite reductase. Hence, combining nitrite with biocides allows more efficient and cost-effective control of sulfate-reducing bacteria.

scholarly journals Design, Synthesis, and Biological Evaluation of Pyridazinones Containing the (2-Fluorophenyl) Piperazine Moiety as Selective MAO-B Inhibitors

Molecules ◽  
2020 ◽  
Vol 25 (22) ◽  
pp. 5371
Author(s):  
Muhammed Çeçen ◽  
Jong Min Oh ◽  
Zeynep Özdemir ◽  
Saliha Ebru Büyüktuncel ◽  
Mehtap Uysal ◽  
...  
Keyword(s):  
Biological Evaluation ◽  
Inhibitory Potency ◽  
Design Synthesis ◽  
Docking Simulations ◽  
Mao B ◽  
Ic50 Value ◽  
Competitive Inhibitors ◽  
Mao A ◽  
Ic50 Values ◽  
Synthesis And Biological Evaluation

Twelve pyridazinones (T1–T12) containing the (2-fluorophenyl) piperazine moiety were designed, synthesized, and evaluated for monoamine oxidase (MAO) -A and -B inhibitory activities. T6 was found to be the most potent MAO-B inhibitor with an IC50 value of 0.013 µM, followed by T3 (IC50 = 0.039 µM). Inhibitory potency for MAO-B was more enhanced by meta bromo substitution (T6) than by para bromo substitution (T7). For para substitution, inhibitory potencies for MAO-B were as follows: -Cl (T3) > -N(CH3)2 (T12) > -OCH3 (T9) > Br (T7) > F (T5) > -CH3 (T11) > -H (T1). T6 and T3 efficiently inhibited MAO-A with IC50 values of 1.57 and 4.19 µM and had the highest selectivity indices (SIs) for MAO-B (120.8 and 107.4, respectively). T3 and T6 were found to be reversible and competitive inhibitors of MAO-B with Ki values of 0.014 and 0.0071, respectively. Moreover, T6 was less toxic to healthy fibroblast cells (L929) than T3. Molecular docking simulations with MAO binding sites returned higher docking scores for T6 and T3 with MAO-B than with MAO-A. These results suggest that T3 and T6 are selective, reversible, and competitive inhibitors of MAO-B and should be considered lead candidates for the treatment of neurodegenerative disorders like Alzheimer’s disease.

scholarly journals The behaviour of leucine aminopeptidase towards thionopeptides

1987 ◽  
Vol 245 (1) ◽  
pp. 285-288 ◽  
Author(s):  
R E Beattie ◽  
D T Elmore ◽  
C H Williams ◽  
D J S Guthrie
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Leucine Aminopeptidase ◽  
Converting Enzyme ◽  
Carboxypeptidase A ◽  
Inhibitory Potency ◽  
Pig Kidney ◽  
Competitive Inhibitors ◽  
Kinetics Of ◽  
Kinetics Of Inhibition

Thionoleucine S-anilide (Leut-anilide), Leut-Gly-OEt and Leut-Phe-OMe were synthesized and shown to be competitive inhibitors of leucine aminopeptidase from pig kidney. The kinetics of inhibition were determined in the presence of leucine 4-methylcoumarin-7-amide as substrate. Although the compounds showed only moderate inhibitory potency, it was found that all were resistant to hydrolysis by the enzyme, in contrast with the reported behaviour of some thionopeptide analogues of substrates for other Zn2+-peptidases such as carboxypeptidase A and angiotensin-converting enzyme.

scholarly journals In Vitro and In Silico Kinetic Studies of Patented 1,7-diEthyl and 1,7-diMethyl Aminoalkanol Derivatives as New Inhibitors of Acetylcholinesterase

2021 ◽  
Vol 23 (1) ◽  
pp. 270
Author(s):  
Błażej Grodner ◽  
Mariola Napiórkowska ◽  
Dariusz Pisklak
Keyword(s):  
Acetylcholinesterase Inhibitors ◽  
Kinetic Studies ◽  
Docking Studies ◽  
Inhibitory Potency ◽  
Main Compound ◽  
Competitive Inhibitors ◽  
Ic50 Values ◽  
Visible Effect ◽  
Derivatives Of

Two aminoalkanol derivatives of 1,7-diEthyl-8,9-diphenyl-4azatricyclo (5.2.1.02,6) dec-8-ene-3,5,10-trione and two derivatives of 1,7-diMethyl-8,9-diphenyl-4-azatricyclo (5.2.1.02.6) dec-8-ene-3,5,10-trione were evaluated in vitro for their inhibition efficacy of acetylcholinesterase. The Km, Vmax, slope angles of Lineweaver–Burk plots, Ki and IC50 values showed that all four aminoalkanol derivatives are competitive inhibitors of acetylcholinesterase whose inhibitory potency depends, to a varying extent, on the nature of the four different substituents present in the main compound structure. Studies have shown that the most potent acetylcholinesterase inhibitors are derivatives containing isopropylamine and/or methyl substituents in their structure. In contrast, dimethylamine and/or ethyl substituents seem to have a weaker, albeit visible, effect on the inhibitory potency of acetylcholinesterase. Additionally, docking studies suggest that studied compounds binds with the peripheral anionic site and not enter into the catalytic pocket due to the presence of the sterically extended substituent.

scholarly journals Nutrient Inputs Stimulate Mercury Methylation by Syntrophs in a Subarctic Peatland

2021 ◽  
Vol 12 ◽  
Author(s):  
Spencer Roth ◽  
Brett A. Poulin ◽  
Zofia Baumann ◽  
Xiao Liu ◽  
Lin Zhang ◽  
...  
Keyword(s):  
Peat Soil ◽  
Metabolic Inhibitors ◽  
Nutrient Inputs ◽  
Sulfate Reducing ◽  
Genetic Potential ◽  
Permafrost Soils ◽  
Input Source ◽  
Thawing Permafrost ◽  
Microbial Groups ◽  
Reducing Bacteria

Climate change dramatically impacts Arctic and subarctic regions, inducing shifts in wetland nutrient regimes as a consequence of thawing permafrost. Altered hydrological regimes may drive changes in the dynamics of microbial mercury (Hg) methylation and bioavailability. Important knowledge gaps remain on the contribution of specific microbial groups to methylmercury (MeHg) production in wetlands of various trophic status. Here, we measured aqueous chemistry, potential methylation rates (kmeth), volatile fatty acid (VFA) dynamics in peat-soil incubations, and genetic potential for Hg methylation across a groundwater-driven nutrient gradient in an interior Alaskan fen. We tested the hypotheses that (1) nutrient inputs will result in increased methylation potentials, and (2) syntrophic interactions contribute to methylation in subarctic wetlands. We observed that concentrations of nutrients, total Hg, and MeHg, abundance of hgcA genes, and rates of methylation in peat incubations (kmeth) were highest near the groundwater input and declined downgradient. hgcA sequences near the input were closely related to those from sulfate-reducing bacteria (SRB), methanogens, and syntrophs. Hg methylation in peat incubations collected near the input source (FPF2) were impacted by the addition of sulfate and some metabolic inhibitors while those down-gradient (FPF5) were not. Sulfate amendment to FPF2 incubations had higher kmeth relative to unamended controls despite no effect on kmeth from addition of the sulfate reduction inhibitor molybdate. The addition of the methanogenic inhibitor BES (25 mM) led to the accumulation of VFAs, but unlike molybdate, it did not affect Hg methylation rates. Rather, the concurrent additions of BES and molybdate significantly decreased kmeth, suggesting a role for interactions between SRB and methanogens in Hg methylation. The reduction in kmeth with combined addition of BES and molybdate, and accumulation of VFA in peat incubations containing BES, and a high abundance of syntroph-related hgcA sequences in peat metagenomes provide evidence for MeHg production by microorganisms growing in syntrophy. Collectively the results suggest that wetland nutrient regimes influence the activity of Hg methylating microorganisms and, consequently, Hg methylation rates. Our results provide key information about microbial Hg methylation and methylating communities under nutrient conditions that are expected to become more common as permafrost soils thaw.

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