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Exploration of Pyrazolo[1,5-a]pyrimidines as Membrane-Bound Pyrophosphatase Inhibitors.

Inhibition of membrane-bound pyrophosphatase (mPPase) with small molecules offer a new approach in the fight against pathogenic protozoan pa … nthetic medicinal chemistry, identifying compounds with low micromolar inhibitory activities in the Thermotoga maritima mPPase test system. We furthermore provide early structure-activity relationships around a new scaffold having a pyrazolo[1,5-a]pyrimidine core. The most promising pyrazolo[1,5-a]pyrimidine congener was further investigated and found to inhibit Plas …

Synthesis of Chiral Acyclic Pyrimidine Nucleoside Analogues from DHAP-Dependent Aldolases.

Dihydroxyacetone phosphate (DHAP)-dependent aldolases catalyze the aldol addition of DHAP to a variety of aldehydes and generate compounds with two stereocenters. This reaction is useful to synthesize chiral acyclic nucleosides, which constitute a well-known class of antiviral drugs currently used. In such compounds, the chirality of the aliphatic chain, which mimics the open pentose residue, is crucial for activity. In this work, three DHAP-dependent aldolases: fructose-1,6-biphosphate aldolase from rabbit muscle, rhanmulose-1-phosphate aldolase from Thermotoga maritima, and fuculose-1-phosph…

Genomic analysis of the F3031 Brazilian purpuric fever clone of Haemophilus influenzae biogroup aegyptius by PCR-based subtractive hybridization.

PCR-based subtractive genome hybridization produced clones harboring inserts present in Brazilian purpuric fever (BPF) prototype strain F3031 but absent in noninvasive Haemophilus influenzae biogroup aegyptius isolate F1947. Some of these inserts have no matches in the GenBank database, while others are similar to genes encoding either known or hypothetical proteins. One insert represents a 2.3-kb locus with similarity to a Thermotoga maritima hypothetical protein, while another is part of a 7.6-kb locus that contains predicted genes encoding hypothetical, phage-related, and carotovoricin Er-l…

Probing the functional tolerance of the b subunit of Escherichia coli ATP synthase for sequence manipulation through a chimera approach.

A dimer of 156-residue b subunits forms the peripheral stator stalk of eubacterial ATP synthase. Dimerization is mediated by a sequence with … iled coils. Replacement of positions 55-110 with corresponding sequences from Bacillus subtilis and Thermotoga maritima b subunits resulted in fully functional chimeras, judged by support of growth on nonfermentable carbon sources. Extension of the T. maritima sequence N-terminally to position 37 or C-terminally to position 124 resulted in slower but significant grow …

Enzymatic synthesis of neotrehalose: Deciphering the β-1 catalytic promiscuity of transglycolytic starch-active enzymes.

Neotrehalose is a naturally occurring, non-reducing, rare disaccharide with promising nutritional, medical, and synthetic applications, but its low natural abundance and complex chemical synthesis limit commercial production. This study evaluated starch-active enzymes as a sustainable route for its synthesis from renewable substrates. A one-step biocatalytic process using starch and/or maltose as glucosyl donors was developed with the hyperthermophilic α-amylase from Thermotoga maritima. Optimized conditions yielded up to 23.2 g neotrehalose/L (11.60 g neotrehalose/100 g initial maltose), amon…

Characterization of the Bacillus anthracis S-layer: cloning and sequencing of the structural gene.

Bacillus anthracis, a gram-positive, spore-forming bacterium, is the etiological agent of anthrax. The gene coding for the S-layer protein ( … milarities with the Acetogenium kivui S-layer protein, the Bacillus brevis middle wall protein, the Thermotoga maritima Omp alpha protein, and the Bacillus thuringiensis S-layer protein. Electron microscopy observations showed that this S-layer is not observed on B. anthracis cells in which sap has been deleted.

Non-complexed four cascade enzyme mixture: simple purification and synergetic co-stabilization.

Cell-free biosystems comprised of synthetic enzymatic pathways would be a promising biomanufacturing platform due to several advantages, suc … riosephosphate isomerase (TIM) from Thermus thermophiles, fructose bisphosphate aldolase (ALD) from Thermotoga maritima, fructose bisphosphatase (FBP) from T. maritima, and phosphoglucose isomerase (PGI) from Clostridium thermocellum. It was found that TIM and ALD were very stable at evaluated temperature so that they were purified by heat precipitation followed by g …

Thermodynamic stability of proteins adapted to extreme temperatures.

Small cold shock proteins from bacteria are model proteins for many biophysical studies as a result of their small size and compact folding. Here we report a stability study of these proteins from the Antarctic psychrophile Pseudoalteromonas haloplanktis, the mesophile Escherichia coli and the hyperthermophile Thermotoga maritima. When analyzed by differential scanning calorimetry, the three proteins unfolded fully reversibly according to a perfect two-state model, which are strict prerequisites to apply equilibrium thermodynamic equations. The parameters obtained by differential scanning calo…

An enzymatic activation of formaldehyde for nucleotide methylation.

Folate enzyme cofactors and their derivatives have the unique ability to provide a single carbon unit at different oxidation levels for the de novo synthesis of amino-acids, purines, or thymidylate, an essential DNA nucleotide. How these cofactors mediate methylene transfer is not fully settled yet, particularly with regard to how the methylene is transferred to the methylene acceptor. Here, we uncovered that the bacterial thymidylate synthase ThyX, which relies on both folate and flavin for activity, can also use a formaldehyde-shunt to directly synthesize thymidylate. Combining biochemical,

Origin of Nitric Oxide Reduction Activity in Flavo-Diiron NO Reductase: Key Roles of the Second Coordination Sphere.

The second coordination sphere constitutes a distinguishing factor in the active site to modulate enzymatic reactivity. To unravel the origin of NO-to-N2 O reduction activity of non-heme diiron enzymes, herein we report a strong second-coordination-sphere interaction between a conserved Tyr197 and the key iron-nitrosyl intermediate of Tm FDP (flavo-diiron protein), which leads to decreased reaction barriers towards N-N formation and N-O cleavage in NO reduction. This finding supports the direct coupling of diiron dinitrosyl as the N-N formation mode in our QM/MM modeling, and reconciles the me

Evaluation of expanded 2-aminobenzothiazole library as inhibitors of a model histidine kinase and virulence suppressors in Pseudomonas aeruginosa.

Bacterial resistance to antibiotics is a rapidly increasing threat to human health. New strategies to combat resistant organisms are despera … the CA domain. We found that these compounds, which inhibit the model histidine kinase, HK853 from Thermotoga maritima, have anti-virulence activities inPseudomonas aeruginosa, reducing motility phenotypes and toxin production associated with the pathogenic functions of this bacterium.

The hyperthermophilic archaeon Thermococcus kodakarensis is resistant to pervasive negative supercoiling activity of DNA gyrase.

In all cells, DNA topoisomerases dynamically regulate DNA supercoiling allowing essential DNA processes such as transcription and replication to occur. How this complex system emerged in the course of evolution is poorly understood. Intriguingly, a single horizontal gene transfer event led to the successful establishment of bacterial gyrase in Archaea, but its emergent function remains a mystery. To better understand the challenges associated with the establishment of pervasive negative supercoiling activity, we expressed the gyrase of the bacterium Thermotoga maritima in a naïve archaeon Ther…

Реконструкция петли антикодона и форма D-стима приводят к конкретному распознаванию ANN-декодирования tRNA для модификации t6A.

N6-threonylcarbamoyladenosine (t6A) — универсальная трансфертная модификация RNA (tRNA), необходимая для перевода. Модификация устанавливается в положение 37 ANN-декодирование tRNAs (N is A, U, G или C) путем переноса треонилкарбамойла из промежуточного пути, катализированного в бактериях комплексом ЦаБД. Несмотря на строгое требование в отношении 36-UAA-38 Последовательность в субстрате tRNS, структурная основа этой специфичности остается неясной. Мы определили крио-эм-структуры "Термотога маритима Т6А" синтазы, связанные с немодифицированной и изначально модифицированной ТRNA с модификацией

Совпадение в аннотации оригинала
N6-threonylcarbamoyladenosine (t6A) is a universal transfer RNA (tRNA) modification essential for translational fidelity. The modification i … structural basis for this specificity has remained unclear. We determined cryo-EM structures of the Thermotoga maritima t6A synthase bound to unmodified and to natively modified tRNA carrying the t6A37 modification, at a nominal resolution of 3.2 Å. In both structures, A37 is positioned in the active site, and the anticodon loop is remodeled into a zig-zag conformati …

ATPase and Protease Domain Movements in the Bacterial AAA+ Protease FtsH Are Driven by Thermal Fluctuations.

AAA+ proteases are essential players in cellular pathways of protein degradation. Elucidating their conformational behavior is key for understanding their reaction mechanism and, importantly, for elaborating our understanding of mutation-induced protease deficiencies. Here, we study the structural dynamics of the Thermotoga maritima AAA+ hexameric ring metalloprotease FtsH (TmFtsH). Using a single-molecule Förster resonance energy transfer approach to monitor ATPase and protease inter-domain conformational changes in real time, we show that TmFtsH-even in the absence of nucleotide-is a highly …

In Vitro One-Pot 3-Hydroxypropanal Production from Cheap C1 and C2 Compounds.

One- or two-carbon (C1 or C2) compounds have been considered attractive substrates because they are inexpensive and abundant. Methanol and e … A was biosynthesized from formaldehyde and acetaldehyde using deoxyribose-5-phosphate aldolase from Thermotoga maritima (DERATma) and cloned and expressed in Escherichia coli for 3-HPA production. Under optimum conditions, DERATma produced 7 mM 3-HPA from 25 mM substrate (formaldehyde and acetaldehyde) for 60 min with 520 mg/L/h productivity. To demonstrate the one-p …

Metal specificity is correlated with two crucial active site residues in Escherichia coli alkaline phosphatase.

Escherichia coli alkaline phosphatase exhibits maximal activity when Zn(2+) fills the M1 and M2 metal sites and Mg(2+) fills the M3 metal site. When other metals replace the zinc and magnesium, the catalytic efficiency is reduced by more than 5000-fold. Alkaline phosphatases from organisms such as Thermotoga maritima and Bacillus subtilis require cobalt for maximal activity and function poorly with zinc and magnesium. Previous studies have shown that the D153H alkaline phosphatase exhibited very little activity in the presence of cobalt, while the K328W and especially the D153H/K328W mutant en…

Leveraging MrkA Display on Self-Assembling Protein Nanoparticles for Enhanced Immune Activation.

Self-assembling protein nanoparticles (NPs) offer a versatile platform to enhance antigen immunogenicity through a multivalent display. Kleb … MrkA to four distinct self-assembling protein nanoparticle scaffolds: Helicobacter pylori ferritin, Thermotoga maritima encapsulin, computationally designed mI3, and Mycobacterium tuberculosis dodecin. The resulting MrkA-NPs were expressed and purified, and their structural integrity was confirmed by electron microscopy, dynamic light scattering, and nuclear magnetic …

Synergic action of an inserted carbohydrate-binding module in a glycoside hydrolase family 5 endoglucanase.

Most known cellulase-associated carbohydrate-binding modules (CBMs) are attached to the N- or C-terminus of the enzyme or are expressed sepa … gth and CBM-deleted MtGlu5 proteins. Conversely, insertion of the CBM from MtGlu5 into TmCel5A from Thermotoga maritima greatly enhanced the substrate affinity of TmCel5A. Bound sugars observed between two tryptophan side chains in the catalytic domains of active full-length and CBM-deleted MtGlu5 suggest an important stacking force. The synergistic action of the cat …

An Aldolase-Based New Pathway for Bioconversion of Formaldehyde and Ethanol into 1,3-Propanediol in Escherichia coli.

Formaldehyde (HCHO) is a reactive one-carbon compound that is interesting for biosynthesis. The assimilation of HCHO depends on the catalysi … . The pathway is successfully demonstrated by elaborating three metabolic modules. First, DERA from Thermotoga maritima was found to be efficient for the aldol condensation and PDO production module. For the module of acetaldehyde supply from ethanol, an alcohol dehydrogenase from Hansenula polymorpha was selected. For the HCHO supply module, the control of HCHO conc …

The molecular architecture of the metalloprotease FtsH.

The ATP-dependent integral membrane protease FtsH is universally conserved in bacteria. Orthologs exist in chloroplasts and mitochondria, where in humans the loss of a close FtsH-homolog causes a form of spastic paraplegia. FtsH plays a crucial role in quality control by degrading unneeded or damaged membrane proteins, but it also targets soluble signaling factors like sigma(32) and lambda-CII. We report here the crystal structure of a soluble FtsH construct that is functional in caseinolytic and ATPase assays. The molecular architecture of this hexameric molecule consists of two rings where t