ABSTRACT NrdR is a universal transcriptional repressor of bacterial genes coding for ribonucleotide reductases (RNRs), essential enzymes that provide DNA building blocks in all living cells. Despite its prevalence, the mechanism has been scarcely studied. We report biochemical, biophysical, and bioinformatical characterization binding sites from two major pathogens phylum Bacillota Listeria monocytogenes Streptococcus pneumoniae . consists Zn‐ribbon domain followed by an ATP‐cone domain....

Escherichia coli possesses class Ia, Ib, and III ribonucleotide reductases (RNR). Under standard laboratory conditions, the aerobic Ia nrdAB RNR genes are well expressed, whereas Ib nrdEF poorly expressed. The is normally expressed under microaerophilic anaerobic conditions. In this paper, we show that E. YbaD protein differentially regulates expression of three sets genes. a homolog Streptomyces NrdR protein. It not essential for growth has been renamed NrdR. Previously, was shown to...

Ribonucleotide reductases (RNRs) are key enzymes in DNA metabolism, with allosteric mechanisms controlling substrate specificity and overall activity. In RNRs, the activity master-switch, ATP-cone, has been found exclusively catalytic subunit. two class I RNR subclasses whose subunit lacks we discovered ATP-cones radical-generating The ATP-cone Leeuwenhoekiella blandensis regulates via quaternary structure induced by binding of nucleotides. ATP induces enzymatically competent dimers, whereas...

NrdR is a bacterial transcriptional repressor consisting of zinc (Zn)‐ribbon domain followed by an ATP‐cone domain. Understanding its mechanism action could aid the design novel antibacterials. binds specifically to two “NrdR boxes” upstream ribonucleotide reductase operons, which Escherichia coli has three: nrdHIEF, nrdDG and nrdAB, in last we identified new box. We show that E. (EcoNrdR) similar binding strength all three sites when loaded with ATP plus deoxyadenosine triphosphate (dATP)...

ABSTRACT Lactobacillus plantarum is an attractive candidate for bioprocessing of lignocellulosic biomass due to its high metabolic variability, including ability ferment both pentoses and hexoses, as well acid tolerance, a quality often utilized in industrial processes. This bacterium grows naturally on biomass; however, it lacks the inherent deconstruct substrates. As first step toward engineering lignocellulose-converting lactobacilli, we have introduced genes coding GH6 cellulase GH11...

The Gram-positive, anaerobic, cellulolytic, thermophile Clostridium (Ruminiclostridium) thermocellum secretes a multi-enzyme system called the cellulosome to solubilize plant cell wall polysaccharides. During saccharolytic process, enzymatic composition of is modulated according type polysaccharide(s) present in environment. C. has set eight alternative RNA polymerase sigma (σ) factors that are activated response extracellular polysaccharides and share sequence similarity Bacillus subtilis...

Non-cellulosomal processive endoglucanase 9I (Cel9I) from Clostridium thermocellum is a modular protein, consisting of family-9 glycoside hydrolase (GH9) catalytic module and two family-3 carbohydrate-binding modules (CBM3c CBM3b), separated by linker regions. GH9 does not show cellulase activity when expressed without CBM3c CBM3b the presence was previously shown to be essential for activity. Physical reassociation independently (containing sequences) restored 60–70% intact Cel9I...

Abstract Ribonucleotide reductase (RNR) is an essential enzyme that catalyzes the synthesis of DNA building blocks in virtually all living cells. NrdR, RNR-specific repressor, controls transcription RNR genes and, often, its own, most bacteria and some archaea. NrdR senses concentration nucleotides through ATP-cone, evolutionarily mobile domain also regulates enzymatic activity many RNRs, while a Zn-ribbon mediates binding to boxes upstream overlapping start site genes. Here, we combine...

Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides and are essential for de novo DNA synthesis repair. Streptomyces spp. contain genes coding two RNRs, either which is sufficient vegetative growth. The class Ia RNR encoded by nrdAB genes, II nrdJ, coexpressed with nrdR. We previously showed that coelicolor nrdR gene encodes a protein, NrdR, represses transcription both sets genes. NrdR member highly conserved family proteins confined...

Ruminococcus albus, a cellulolytic bacterium, is critical member of the rumen community. albus lacks classical cellulosome complex, but it possesses unique family 37 carbohydrate-binding module (CBM37), which integrated into variety carbohydrate-active enzymes. We developed potential molecular tool for functional phylotyping R. population in rumen, based on variable region cel48A gene. encodes single copy CBM37-associated 48 glycoside hydrolase all known strains this bacterium. A segment...

Abstract NrdR is a bacterial transcriptional repressor consisting of Zn-ribbon domain followed by an ATP-cone domain. Understanding its mechanism action could aid the design novel antibacterials. binds specifically to two “NrdR boxes” upstream ribonucleotide reductase operons, which Escherichia coli has three: nrdHIEF, nrdDG and nrdAB, where we identified new box. We show that E. (EcoNrdR) similar binding strength all three sites when loaded with ATP plus dATP or equivalent diphosphate...

Class I ribonucleotide reductase (RNR) consists of a catalytic subunit (NrdA) and radical-generating (NrdB) that together catalyze reduction ribonucleotides to their corresponding deoxyribonucleotides. NrdB from the firmicute Facklamia ignava is unique fusion protein with N-terminal add-ons glutaredoxin (Grx) domain followed by an ATP-binding domain, ATP cone. Grx, usually encoded separately RNR operon, known reductant. We show fused Grx functions as efficient reductant F. class via common...

Ribonucleotide reductases (RNRs) are essential enzymes in all living cells, providing the only known de novo pathway for biosynthesis of deoxyribonucleotides (dNTPs), immediate precursors DNA synthesis and repair. RNRs catalyze controlled reduction four ribonucleotides to maintain a balanced pool dNTPs during cell cycle. Streptomyces species contain genes, nrdAB nrdJ, coding oxygen-dependent class I oxygen-independent II RNRs, either which is sufficient vegetative growth. Both sets genes...

Background Ruminococcus flavefaciens is one of the predominant fiber-degrading bacteria found in rumen herbivores. Bioinformatic analysis recently sequenced genome indicated that this bacterium produces most intricate cellulosome systems known to date. A distinct ORF, encoding for a multi-modular protein, RflaF_05439, was discovered during mining sequence. It composed two tandem modules currently undefined function share 45% identity and C-terminal X-dockerin modular dyad. Gaining insight...

ABSTRACT We report the single-contig genome sequence of anaerobic, mesophilic, cellulolytic bacterium, Bacteroides cellulosolvens . The bacterium produces a particularly elaborate cellulosome system, wherein types cohesin-dockerin interactions are opposite other known systems: cell-surface attachment is thus mediated via type-I interactions, whereas enzymes integrated type-II interactions.

Electroendocytosis involves the exposure of cells to pulsed low electric field and is emerging as a complementary method electroporation for incorporation macromolecules into cells. The present study explores underlying mechanism electroendocytosis its dependence on electrochemical byproducts formed at electrode interface. Cell suspensions were exposed in partitioned device where are spatially restricted relative electrodes. cellular uptake dextran-FITC was analyzed by flow cytometery...

Outside of the photosynthetic machinery, high-valent manganese cofactors are rare in biology. It was proposed that a recently discovered subclass ribonucleotide reductase (RNR), class Id, is dependent on Mn2(IV,III) cofactor for catalysis. Class I RNRs consist substrate-binding component (NrdA) and metal-containing radical-generating (NrdB). Herein we utilize combination EPR spectroscopy enzyme assays to underscore enzymatic relevance Id NrdB from Facklamia ignava. Once formed, confers...

Cellulolytic clostridia use a highly efficient cellulosome system to degrade polysaccharides. To regulate genes encoding enzymes of the multi-enzyme complex, certain contain alternative sigma I (σI ) factors that have cognate membrane-associated anti-σI (RsgIs) which act as polysaccharide sensors. In this work, we analyzed structure-function relationship extracellular sensory elements Clostridium (Ruminiclostridium) thermocellum and clariflavum (RsgI3 RsgI4, respectively). These were...

Ribonucleotide reductase (RNR) is an essential enzyme with a complex mechanism of allosteric regulation found in nearly all living organisms. Class I RNRs are composed two proteins, large α-subunit (R1) and smaller β-subunit (R2) that exist as homodimers, combine to form active heterotetramer. Aquifex aeolicus hyperthermophilic bacterium unusual RNR encoding 346-residue intein the DNA sequence its R2 subunit. We present first structures A. R1 (AaR1 AaR2, respectively) proteins well...

The essential enzyme ribonucleotide reductase (RNR) is highly regulated both at the level of overall activity and substrate specificity. Studies class I, aerobic RNRs have shown that downregulated by binding dATP to a small domain known as ATP-cone often found N-terminus RNR subunits, causing oligomerization prevents formation necessary α 2 β complex between catalytic (α ) radical generating (β subunits. In some relatively rare organisms with subclass NrdAi, subunit rather than more commonly...

A small, nucleotide-binding domain, the ATP-cone, is found at N-terminus of most ribonucleotide reductase (RNR) catalytic subunits. By binding ATP or dATP it regulates enzyme activity all classes RNR. Functional and structural work on aerobic RNRs has revealed a plethora ways in which inhibits by inducing oligomerization preventing productive radical transfer from one subunit to active site other. Anaerobic RNRs, other hand, store stable glycyl next basis for their dATP-dependent inhibition...