Browse
Submit Data
Databases
API
Help

Dataset Information

16 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Structural and Biochemical Investigation of Class I Ribonucleotide Reductase from the Hyperthermophile Aquifex aeolicus.

ABSTRACT: 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 of two proteins, a large α-subunit (R1) and a smaller β-subunit (R2) that exist as homodimers, that combine to form an active heterotetramer. Aquifex aeolicus is a hyperthermophilic bacterium with an unusual RNR encoding a 346-residue intein in the DNA sequence encoding its R2 subunit. We present the first structures of the A. aeolicus R1 and R2 (AaR1 and AaR2, respectively) proteins as well as the biophysical and biochemical characterization of active and inactive A. aeolicus RNR. While the active oligomeric state and activity regulation of A. aeolicus RNR are similar to those of other characterized RNRs, the X-ray crystal structures also reveal distinct features and adaptations. Specifically, AaR1 contains a β-hairpin hook structure at the dimer interface, which has an interesting π-stacking interaction absent in other members of the NrdAh subclass, and its ATP cone houses two ATP molecules. We determined structures of two AaR2 proteins: one purified from a construct lacking the intein (AaR2) and a second purified from a construct including the intein sequence (AaR2_genomic). These structures in the context of metal content analysis and activity data indicate that AaR2_genomic displays much higher iron occupancy and activity compared to AaR2, suggesting that the intein is important for facilitating complete iron incorporation, particularly in the Fe2 site of the mature R2 protein, which may be important for the survival of A. aeolicus in low-oxygen environments.

SUBMITTER: Rehling D

PROVIDER: S-EPMC8772380 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Ligation reaction specificities of an NAD(+)-dependent DNA ligase from the hyperthermophile Aquifex aeolicus.

Project description:An NAD(+)-dependent DNA ligase from the hyperthermophilic bacterium Aquifex aeolicus was cloned, expressed in Escherichia coli and purified to homogeneity. The enzyme is most active in slightly alkaline pH conditions with either Mg(2+)or Mn(2+)as the metal cofactor. Ca(2+)and Ni(2+)mainly support formation of DNA-adenylate intermediates. The catalytic cycle is characterized by a low k (cat)value of 2 min(-1)with concomitant accumulation of the DNA - adenylate intermediate when Mg(2+)is used as the metal cofactor. The ligation rates of matched substrates vary by up to 4-fold, but exhibit a general trend of T/A < or = G/C < C/G < A/T on both the 3'- and 5'-side of the nick. Consistent with previous studies on Thermus ligases, this Aquifex ligase exhibits greater discrimination against a mismatched base pair on the 3'-side of the nick junction. The requirement of 3' complementarity for a ligation reaction is reaffirmed by results from 1 nt insertions on either the 3'- or 5'-side of the nick. Furthermore, most of the unligatable 3' mismatched base pairs prohibit formation of the DNA-adenylate intermediate, indicating that the substrate adenylation step is also a control point for ligation fidelity. Unlike previously studied ATP ligases, gapped substrates cannot be ligated and intermediate accumulation is minimal, suggesting that complete elimination of base pair complementarity on one side of the nick affects substrate adenylation on the 5'-side of the nick junction. Relationships among metal cofactors, ligation products and intermediate, and ligation fidelity are discussed.

| S-EPMC111035 | biostudies-literature

Crystal structure of THEP1 from the hyperthermophile Aquifex aeolicus: a variation of the RecA fold.

Project description:BackgroundaaTHEP1, the gene product of aq_1292 from Aquifex aeolicus, shows sequence homology to proteins from most thermophiles, hyperthermophiles, and higher organisms such as man, mouse, and fly. In contrast, there are almost no homologous proteins in mesophilic unicellular microorganisms. aaTHEP1 is a thermophilic enzyme exhibiting both ATPase and GTPase activity in vitro. Although annotated as a nucleotide kinase, such an activity could not be confirmed for aaTHEP1 experimentally and the in vivo function of aaTHEP1 is still unknown.ResultsHere we report the crystal structure of selenomethionine substituted nucleotide-free aaTHEP1 at 1.4 A resolution using a multiple anomalous dispersion phasing protocol. The protein is composed of a single domain that belongs to the family of 3-layer (alpha/beta/alpha)-structures consisting of nine central strands flanked by six helices. The closest structural homologue as determined by DALI is the RecA family. In contrast to the latter proteins, aaTHEP1 possesses an extension of the beta-sheet consisting of four additional beta-strands.ConclusionWe conclude that the structure of aaTHEP1 represents a variation of the RecA fold. Although the catalytic function of aaTHEP1 remains unclear, structural details indicate that it does not belong to the group of GTPases, kinases or adenosyltransferases. A mainly positive electrostatic surface indicates that aaTHEP1 might be a DNA/RNA modifying enzyme. The resolved structure of aaTHEP1 can serve as paradigm for the complete THEP1 family.

| S-EPMC1079890 | biostudies-literature

Aquifex aeolicus

Project description:Hyperthermophilic bacterium

| PRJNA35137 | ENA

Structural basis for activation of class Ib ribonucleotide reductase.

Project description:The class Ib ribonucleotide reductase of Escherichia coli can initiate reduction of nucleotides to deoxynucleotides with either a Mn(III)2-tyrosyl radical (Y•) or a Fe(III)2-Y• cofactor in the NrdF subunit. Whereas Fe(III)2-Y• can self-assemble from Fe(II)2-NrdF and O2, activation of Mn(II)2-NrdF requires a reduced flavoprotein, NrdI, proposed to form the oxidant for cofactor assembly by reduction of O2. The crystal structures reported here of E. coli Mn(II)2-NrdF and Fe(II)2-NrdF reveal different coordination environments, suggesting distinct initial binding sites for the oxidants during cofactor activation. In the structures of Mn(II)2-NrdF in complex with reduced and oxidized NrdI, a continuous channel connects the NrdI flavin cofactor to the NrdF Mn(II)2 active site. Crystallographic detection of a putative peroxide in this channel supports the proposed mechanism of Mn(III)2-Y• cofactor assembly.

| S-EPMC3020666 | biostudies-literature

The elusive third subunit IIa of the bacterial B-type oxidases: the enzyme from the hyperthermophile Aquifex aeolicus.

Project description:The reduction of molecular oxygen to water is catalyzed by complicated membrane-bound metallo-enzymes containing variable numbers of subunits, called cytochrome c oxidases or quinol oxidases. We previously described the cytochrome c oxidase II from the hyperthermophilic bacterium Aquifex aeolicus as a ba(3)-type two-subunit (subunits I and II) enzyme and showed that it is included in a supercomplex involved in the sulfide-oxygen respiration pathway. It belongs to the B-family of the heme-copper oxidases, enzymes that are far less studied than the ones from family A. Here, we describe the presence in this enzyme of an additional transmembrane helix "subunit IIa", which is composed of 41 amino acid residues with a measured molecular mass of 5105 Da. Moreover, we show that subunit II, as expected, is in fact longer than the originally annotated protein (from the genome) and contains a transmembrane domain. Using Aquifex aeolicus genomic sequence analyses, N-terminal sequencing, peptide mass fingerprinting and mass spectrometry analysis on entire subunits, we conclude that the B-type enzyme from this bacterium is a three-subunit complex. It is composed of subunit I (encoded by coxA(2)) of 59000 Da, subunit II (encoded by coxB(2)) of 16700 Da and subunit IIa which contain 12, 1 and 1 transmembrane helices respectively. A structural model indicates that the structural organization of the complex strongly resembles that of the ba(3) cytochrome c oxidase from the bacterium Thermus thermophilus, the IIa helical subunit being structurally the lacking N-terminal transmembrane helix of subunit II present in the A-type oxidases. Analysis of the genomic context of genes encoding oxidases indicates that this third subunit is present in many of the bacterial oxidases from B-family, enzymes that have been described as two-subunit complexes.

| S-EPMC3128077 | biostudies-literature

Biochemical Characterization of the Split Class II Ribonucleotide Reductase from Pseudomonas aeruginosa.

Project description:The opportunistic pathogen Pseudomonas aeruginosa can grow under both aerobic and anaerobic conditions. Its flexibility with respect to oxygen load is reflected by the fact that its genome encodes all three existing classes of ribonucleotides reductase (RNR): the oxygen-dependent class I RNR, the oxygen-indifferent class II RNR, and the oxygen-sensitive class III RNR. The P. aeruginosa class II RNR is expressed as two separate polypeptides (NrdJa and NrdJb), a unique example of a split RNR enzyme in a free-living organism. A split class II RNR is also found in a few closely related ?-Proteobacteria. We have characterized the P. aeruginosa class II RNR and show that both subunits are required for formation of a biologically functional enzyme that can sustain vitamin B12-dependent growth. Binding of the B12 coenzyme as well as substrate and allosteric effectors resides in the NrdJa subunit, whereas the NrdJb subunit mediates efficient reductive dithiol exchange during catalysis. A combination of activity assays and activity-independent methods like surface plasmon resonance and gas phase electrophoretic macromolecule analysis suggests that the enzymatically active form of the enzyme is a (NrdJa-NrdJb)2 homodimer of heterodimers, and a combination of hydrogen-deuterium exchange experiments and molecular modeling suggests a plausible region in NrdJa that interacts with NrdJb. Our detailed characterization of the split NrdJ from P. aeruginosa provides insight into the biochemical function of a unique enzyme known to have central roles in biofilm formation and anaerobic growth.

| S-EPMC4520616 | biostudies-literature

Structure of a class II TrmH tRNA-modifying enzyme from Aquifex aeolicus.

Project description:Biological RNAs contain a variety of post-transcriptional modifications that facilitate their efficient function in the cellular environment. One of the two most common forms of modification is methylation of the 2'-hydroxyl group of the ribose sugar, which is performed by a number of S-adenosylmethionine (SAM) dependent methyltransferases. In bacteria, many of these modifications in tRNA and rRNA are carried out by the alpha/beta-knot superfamily of enzymes, whose SAM-binding pocket is created by a characteristic deep trefoil knot. TrmH, an enzyme found throughout all three kingdoms of life, modifies the universally conserved guanosine 18 position of tRNA. The crystal structure of TrmH from the thermophilic bacterium Aquifex aeolicus has been determined at 1.85 A resolution using data collected from a synchrotron-radiation source. The protein reveals a fold typical of members of the SpoU clan of proteins, a subfamily of the alpha/beta-knot superfamily, with alpha-helical extensions at the N- and C-termini that are likely to be involved in tRNA binding.

| S-EPMC1952360 | biostudies-literature

Aquifex aeolicus VF5

Project description:Hyperthermophilic organism that is one of the deepest branching organism

| PRJNA215 | ENA

Structural mechanism of GAF-regulated σ(54) activators from Aquifex aeolicus.

Project description:The σ subunits of bacterial RNA polymerase occur in many variant forms and confer promoter specificity to the holopolymerase. Members of the σ(54) family of σ subunits require the action of a 'transcriptional activator' protein to open the promoter and initiate transcription. The activator proteins undergo regulated assembly from inactive dimers to hexamers that are active ATPases. These contact σ(54) directly and, through ATP hydrolysis, drive a conformational change that enables promoter opening. σ(54) activators use several different kinds of regulatory domains to respond to a wide variety of intracellular signals. One common regulatory module, the GAF domain, is used by σ(54) activators to sense small-molecule ligands. The structural basis for GAF domain regulation in σ(54) activators has not previously been reported. Here, we present crystal structures of GAF regulatory domains for Aquifex aeolicus σ(54) activators NifA-like homolog (Nlh)2 and Nlh1 in three functional states-an 'open', ATPase-inactive state; a 'closed', ATPase-inactive state; and a 'closed', ligand-bound, ATPase-active state. We also present small-angle X-ray scattering data for Nlh2-linked GAF-ATPase domains in the inactive state. These GAF domain dimers regulate σ(54) activator proteins by holding the ATPase domains in an inactive dimer conformation. Ligand binding of Nlh1 dramatically remodels the GAF domain dimer interface, disrupting the contacts with the ATPase domains. This mechanism has strong parallels to the response to phosphorylation in some two-component regulated σ(54) activators. We describe a structural mechanism of GAF-mediated enzyme regulation that appears to be conserved among humans, plants, and bacteria.

| S-EPMC3544215 | biostudies-literature

Dihydroorotase from the hyperthermophile Aquifex aeolicus is activated by stoichiometric association with aspartate transcarbamoylase and forms a one-pot reactor for pyrimidine biosynthesis.

Project description:In prokaryotes, the first three enzymes in pyrimidine biosynthesis, carbamoyl phosphate synthetase (CPS), aspartate transcarbamoylase (ATC), and dihydroorotase (DHO), are commonly expressed separately and either function independently (Escherichia coli) or associate into multifunctional complexes (Aquifex aeolicus). In mammals the enzymes are expressed as a single polypeptide chain (CAD) in the order CPS-DHO-ATC and associate into a hexamer. This study presents the three-dimensional structure of the noncovalent hexamer of DHO and ATC from the hyperthermophile A. aeolicus at 2.3 A resolution. It is the first structure of any multienzyme complex in pyrimidine biosynthesis and is a possible model for the core of mammalian CAD. The structure has citrate, a near isosteric analogue of carbamoyl aspartate, bound to the active sites of both enzymes. Three active site loops that are intrinsically disordered in the free, inactive DHO are ordered in the complex. The reorganization also changes the peptide bond between Asp153, a ligand of the single zinc atom in DHO, and Gly154, to the rare cis conformation. In the crystal structure, six DHO and six ATC chains form a hollow dodecamer, in which the 12 active sites face an internal reaction chamber that is approximately 60 A in diameter and connected to the cytosol by narrow tunnels. The entrances and the interior of the chamber are both electropositive, which suggests that the architecture of this nanoreactor modifies the kinetics of the bisynthase, not only by steric channeling but also by preferential escape of the product, dihydroorotase, which is less negatively charged than its precursors, carbamoyl phosphate, aspartate, or carbamoyl aspartate.

| S-EPMC3863388 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data