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Current Opinion in Structural Biology Oct 2022Enzymes that use thiamin diphosphate (ThDP), the biologically active derivative of vitamin B1, as a cofactor play important roles in cellular metabolism in all domains... (Review)
Review
Enzymes that use thiamin diphosphate (ThDP), the biologically active derivative of vitamin B1, as a cofactor play important roles in cellular metabolism in all domains of life. The analysis of ThDP enzymes in the past decades have provided a general framework for our understanding of enzyme catalysis of this protein family. In this review, we will discuss recent advances in the field that include the observation of "unusual" reactions and reaction intermediates that highlight the chemical versatility of the thiamin cofactor. Further topics cover the structural basis of cooperativity of ThDP enzymes, novel insights into the mechanism and structure of selected enzymes, and the discovery of "superassemblies" as reported, for example, acetohydroxy acid synthase. Finally, we summarize recent findings in the structural organisation and mode of action of 2-keto acid dehydrogenase multienzyme complexes and discuss future directions of this exciting research field.
Topics: Acetolactate Synthase; Diphosphates; Multienzyme Complexes; Oxidoreductases; Thiamine; Thiamine Pyrophosphate
PubMed: 35988322
DOI: 10.1016/j.sbi.2022.102441 -
Chemical Communications (Cambridge,... Mar 2022The biosynthesis of 2-methylisoborneol was reconstituted by elongation of dimethylallyl diphosphate (DMAPP) with ()- and ()-2-methylisopentenyl diphosphate (2-Me-IPP)...
The biosynthesis of 2-methylisoborneol was reconstituted by elongation of dimethylallyl diphosphate (DMAPP) with ()- and ()-2-methylisopentenyl diphosphate (2-Me-IPP) using farnesyl diphosphate synthase (FPPS), followed by terpene cyclisation. The stereochemical course of the FPPS reaction was studied in detail using stereoselectively deuterated 2-Me-IPP isotopomers.
Topics: Camphanes; Diphosphates; Geranyltranstransferase; Terpenes
PubMed: 35262160
DOI: 10.1039/d2cc00636g -
Chembiochem : a European Journal of... May 2022The structural diversity of terpenes is particularly notable and many studies are carried out to increase it further. In the terpene biosynthetic pathway this diversity... (Review)
Review
The structural diversity of terpenes is particularly notable and many studies are carried out to increase it further. In the terpene biosynthetic pathway this diversity is accessible from only two common precursors, i. e. isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Methods recently developed (e. g. the Terpene Mini Path) have allowed DMAPP and IPP to be obtained from a two-step enzymatic conversion of industrially available isopentenol (IOH) and dimethylallyl alcohol (DMAOH) into their corresponding diphosphates. Easily available IOH and DMAOH analogues then offer quick access to modified terpenoids thus avoiding the tedious chemical synthesis of unnatural diphosphates. The aim of this minireview is to cover the literature devoted to the use of these analogues for widening the accessible terpene chemical space.
Topics: Biosynthetic Pathways; Diphosphates; Hemiterpenes; Organophosphorus Compounds; Terpenes
PubMed: 34905641
DOI: 10.1002/cbic.202100642 -
FEMS Microbiology Letters Jan 2023Recycling of undecaprenol pyrophosphate is critical to regenerate the pool of undecaprenol monophosphate required for cell wall biosynthesis. Undecaprenol pyrophosphate...
Recycling of undecaprenol pyrophosphate is critical to regenerate the pool of undecaprenol monophosphate required for cell wall biosynthesis. Undecaprenol pyrophosphate is dephosphorylated by membrane-associated undecaprenyl pyrophosphate phosphatases such as UppP or type 2 Phosphatidic Acid Phosphatases (PAP2) and then transferred across the cytoplasmic membrane by Und-P flippases such as PopT (DUF368-containing protein) or UptA (a DedA family protein). While the deletion of uppP in S. pneumoniae has been reported to increase susceptibility to bacitracin and reduce infectivity in a murine infection model, the presence of PAP2 family proteins or Und-P flippases and their potential interplay with UppP in S. pneumoniae remained unknown. In this report, we identified two PAP2 family proteins and a DUF368-containing protein and investigated their roles together with that of UppP in cell growth, cell morphology and susceptibility to bacitracin in S. pneumoniae. Our results suggest that the undecaprenol monophosphate recycling pathway in S. pneumoniae could result from a functional redundancy between UppP, the PAP2-family protein Spr0434 and the DUF368-containing protein Spr0889.
Topics: Mice; Animals; Bacitracin; Streptococcus pneumoniae; Diphosphates
PubMed: 37849218
DOI: 10.1093/femsle/fnad109 -
Journal of Medicinal Chemistry Sep 2023We report on the synthesis and evaluation of three different nucleotide prodrug systems: (i) nucleoside triphosphate analogues in which the γ-phosph(on)ate has two...
We report on the synthesis and evaluation of three different nucleotide prodrug systems: (i) nucleoside triphosphate analogues in which the γ-phosph(on)ate has two different lipophilic nonbioreversible alkyl residues with d4TDP as the released nucleotide analogue; (ii) nucleoside diphosphate analogues bearing a bioreversible and a stable β-alkyl group; or (iii) nucleoside diphosphate analogues bearing two nonhydrolysable lipophilic alkyl moieties. The delivery of d4TDP (for the triphosphate precursor) and d4TMP (for the diphosphate precursor) was demonstrated in CD4 T-lymphocyte CEM cell extracts as well as in phosphate buffer saline (PBS). In primer extension assay, we found that γ-dialkylated d4TTP derivatives and d4TDP were accepted as substrates by HIV-RT. Several of these compounds were observed to be extremely active against HIV-1/2 replication in HIV-infected cells. A more than 45,000-fold increase in the anti-HIV activity was detected for compound as compared to the parent d4T which results in a selectivity index value of 37,000.
Topics: Diphosphates; Nucleosides; Polyphosphates; Nucleotides
PubMed: 37647547
DOI: 10.1021/acs.jmedchem.3c00755 -
Methods in Enzymology 2020The specific non-invasive control of intracellular signaling events requires advanced tools that enter cells by diffusion and are controllable by light. Here, we detail...
The specific non-invasive control of intracellular signaling events requires advanced tools that enter cells by diffusion and are controllable by light. Here, we detail the synthesis and application of membrane-permeant caged inositol pyrophosphates with respect to cell entry and cell distribution. We recently published the synthesis of these tools as well as their effect on PH-domain localization in HeLa cells and oscillations of the intracellular calcium concentration in β-cells, which are known to drive insulin secretion. In this chapter, we discuss the possibilities and limitations when using cell-penetrating inositol pyrophosphates. We provide a detailed protocol for the application in live mouse β-cells and we discuss the image analysis needed for following effects on calcium signaling.
Topics: Calcium; Diphosphates; HeLa Cells; Humans; Inositol Phosphates; Signal Transduction
PubMed: 32713537
DOI: 10.1016/bs.mie.2020.04.036 -
Cell Biochemistry and Biophysics Dec 2022Phosphoribosyl pyrophosphate synthetase-1 (PRPS-1; EC 2.7.6.1.) catalyzes the binding of phosphate-group to ribose 5-phosphate, forming the...
Phosphoribosyl pyrophosphate synthetase-1 (PRPS-1; EC 2.7.6.1.) catalyzes the binding of phosphate-group to ribose 5-phosphate, forming the 5-phosphoribosyl-1-pyrophosphate, which is necessary for the salvage pathways of purine and pyrimidine, pyridine nucleotide cofactors - NAD and NADP, the amino acids histidine and tryptophan biosynthesis. We aimed to investigate the impact of the different effectors on the activity of PRPS-1, to check the activity of the enzyme in vitro in a wide range of pHs and investigate some structural essentials of the enzyme, isolated from brain and liver. Molecular docking analyses were used to delineate the essentials of PRPS-1 structure, to find out the existence of enzyme effectors. Previously created by us kit was used for determination of the activity of PRPS-1 based on the formation of the inorganic phosphates (λ = 700 nm, Cary 60, Agilent, USA). Effectors impact on the activity of PRPS-1 was evaluated. In silico results of the effectors were later proven by in vitro experiments. For the first time biochemical essentials, including the existence of the multiple pockets, involvement of the amino acids into the processes of interactions with the effectors, evolutional of the sequence conservation, tissue depended V differences were identified.
Topics: Diphosphates; Histidine; Molecular Docking Simulation; NAD; NADP; Nucleotides; Phosphates; Phosphoribosyl Pyrophosphate; Purines; Pyridines; Pyrimidines; Ribose-Phosphate Pyrophosphokinase; Tryptophan
PubMed: 36201097
DOI: 10.1007/s12013-022-01104-1 -
Metabolism: Clinical and Experimental May 2021It is well-established that mitochondria are the powerhouses of the cell, producing adenosine triphosphate (ATP), the universal energy currency. However, the most... (Review)
Review
It is well-established that mitochondria are the powerhouses of the cell, producing adenosine triphosphate (ATP), the universal energy currency. However, the most significant strengths of the electron transport chain (ETC), its intricacy and efficiency, are also its greatest downfalls. A reliance on metal complexes (FeS clusters, hemes), lipid moities such as cardiolipin, and cofactors including alpha-lipoic acid and quinones render oxidative phosphorylation vulnerable to environmental toxins, intracellular reactive oxygen species (ROS) and fluctuations in diet. To that effect, it is of interest to note that temporal disruptions in ETC activity in most organisms are rarely fatal, and often a redundant number of failsafes are in place to permit continued ATP production when needed. Here, we highlight the metabolic reconfigurations discovered in organisms ranging from parasitic Entamoeba to bacteria such as pseudomonads and then complex eukaryotic systems that allow these species to adapt to and occasionally thrive in harsh environments. The overarching aim of this review is to demonstrate the plasticity of metabolic networks and recognize that in times of duress, life finds a way.
Topics: Adenosine Triphosphate; Animals; Basic Helix-Loop-Helix Transcription Factors; Citric Acid Cycle; Diphosphates; Electron Transport; Glycolysis; Heat-Shock Proteins; Humans; Microbiota; Mitochondria; Oxidative Stress; Phosphorylation
PubMed: 33631145
DOI: 10.1016/j.metabol.2021.154733 -
The American Journal of Pathology May 2022Pathologic soft tissue calcification can occur in both genetic and acquired clinical conditions, causing significant morbidity and mortality. Although the... (Review)
Review
Pathologic soft tissue calcification can occur in both genetic and acquired clinical conditions, causing significant morbidity and mortality. Although the pathomechanisms of pathologic calcification are poorly understood, major progress has been made in recent years in defining the underlying genetic defects in Mendelian disorders of ectopic calcification. This review presents an overview of the pathophysiology of five monogenic disorders of pathologic calcification: pseudoxanthoma elasticum, generalized arterial calcification of infancy, arterial calcification due to deficiency of CD73, ankylosis, and progeria. These hereditary disorders, caused by mutations in genes encoding ATP binding cassette subfamily C member 6, ectonucleotide pyrophosphatase/phosphodiesterase 1, CD73, progressive ankylosis protein, and lamin A/C proteins, respectively, are inorganic pyrophosphate (PPi) deficiency syndromes with reduced circulating levels of PPi, the principal physiologic inhibitor of calcium hydroxyapatite deposition in soft connective tissues. In addition to genetic diseases, PPi deficiency has been encountered in acquired clinical conditions accompanied by pathologic calcification. Because specific and effective treatments are lacking for pathologic calcification, the unifying finding of PPi deficiency suggests that PPi-targeted therapies may be beneficial to counteract pathologic soft tissue calcification in both genetic and acquired diseases.
Topics: Ankylosis; Calcinosis; Choristoma; Diphosphates; Humans; Pseudoxanthoma Elasticum; Syndrome; Vascular Calcification
PubMed: 35182493
DOI: 10.1016/j.ajpath.2022.01.012 -
Methods in Enzymology 2023Isoprenoids in plants are synthesized following the plastidial methylerythritol-4-phosphate (MEP) pathway or the mevalonate pathway localized to the cytosol and...
Isoprenoids in plants are synthesized following the plastidial methylerythritol-4-phosphate (MEP) pathway or the mevalonate pathway localized to the cytosol and peroxisomes. Isoprenyl-diphosphates (isoprenyl-PP) are important intermediates for the synthesis of chlorophyll, carotenoids, sterols, and other isoprenoids in plants. The quantification of isoprenyl-PP is challenging due to the amphipathic structure, the low abundance, and the susceptibility to hydrolysis during extraction and storage. Different methods for the measurement of isoprenyl-phosphates have been developed. Isoprenyl-phosphates can be measured after radioactive labeling or after derivatization. Liquid chromatography-mass spectrometry (LC-MS) methods provide enhanced sensitivity, but still require the extraction from large amounts of sample material. In the protocol presented here, the monophosphates and diphosphates of farnesol, geranylgeraniol and phytol are isolated from plant material with an isopropanol-containing buffer and quantified by LC-MS using citronellyl-P and citronellyl-PP as internal standards. With a low limit of detection for phytyl-P, geranylgeranyl-P, phytyl-PP, and geranylgeranyl-PP, isoprenyl-phosphates can be accurately measured in Arabidopsis leaves or seeds starting with only 20mg of fresh weight.
Topics: Diphosphates; Mass Spectrometry; Terpenes; Chromatography, Liquid; Plants; Arabidopsis
PubMed: 37087186
DOI: 10.1016/bs.mie.2022.08.026