Evolution of Ribosomal Protein S14 Demonstrated by the Reconstruction of Chimeric Ribosomes in Bacillus subtilis.

Ribosomal protein S14 can be classified into three types. The first, the C+ type has a Zn binding motif and is ancestral. The second and third are the C- short and C- long types, neither of which contain a Zn binding motif and which are ca.

ATP-binding affinity of the ε subunit of thermophilic F-ATPase under label-free conditions.

The ε subunits of several bacterial F-ATPases bind ATP. ATP binding to the ε subunit has been shown to be involved in the regulation of F-ATPase from thermophilic sp. PS3 (TF).

C-terminal regulatory domain of the ε subunit of F F ATP synthase enhances the ATP-dependent H pumping that is involved in the maintenance of cellular membrane potential in Bacillus subtilis.

The ε subunit of F F -ATPase/synthase (F F ) plays a crucial role in regulating F F activity. To understand the physiological significance of the ε subunit-mediated regulation of F F in Bacillus subtilis, we constructed and characterized a mutant harboring a deletion in the C-terminal regulatory domain of the ε subunit (ε ). Analyses using inverted membrane vesicles revealed that the ε mutation decreased ATPase activity and the ATP-dependent H -pumping activity of F F .

Magnesium Suppresses Defects in the Formation of 70S Ribosomes as Well as in Sporulation Caused by Lack of Several Individual Ribosomal Proteins.

Individually, the ribosomal proteins L1, L23, L36, and S6 are not essential for cell proliferation of , but the absence of any one of these ribosomal proteins causes a defect in the formation of the 70S ribosomes and a reduced growth rate. In mutant strains individually lacking these ribosomal proteins, the cellular Mg content was significantly reduced. The deletion of YhdP, an exporter of Mg, and overexpression of MgtE, the main importer of Mg, increased the cellular Mg content and restored the formation of 70S ribosomes in these mutants.

Mechanistic Insights into the Activation of Soluble Guanylate Cyclase by Carbon Monoxide: A Multistep Mechanism Proposed for the BAY 41-2272 Induced Formation of 5-Coordinate CO-Heme.

Soluble guanylate cyclase (sGC) is a heme-containing enzyme that catalyzes cGMP production upon sensing NO. While the CO adduct, sGC-CO, is much less active, the allosteric regulator BAY 41-2272 stimulates the cGMP productivity to the same extent as that of sGC-NO. The stimulatory effect has been thought to be likely associated with Fe-His bond cleavage leading to 5-coordinate CO-heme, but the detailed mechanism remains unresolved.

Essential Role of the ε Subunit for Reversible Chemo-Mechanical Coupling in F-ATPase.

F-ATPase is a rotary motor protein driven by ATP hydrolysis. Among molecular motors, F exhibits unique high reversibility in chemo-mechanical coupling, synthesizing ATP from ADP and inorganic phosphate upon forcible rotor reversal. The ε subunit enhances ATP synthesis coupling efficiency to > 70% upon rotation reversal.

The structural basis of a high affinity ATP binding ε subunit from a bacterial ATP synthase.

The ε subunit from bacterial ATP synthases functions as an ATP sensor, preventing ATPase activity when the ATP concentration in bacterial cells crosses a certain threshold. The R103A/R115A double mutant of the ε subunit from thermophilic Bacillus PS3 has been shown to bind ATP two orders of magnitude stronger than the wild type protein. We use molecular dynamics simulations and free energy calculations to derive the structural basis of the high affinity ATP binding to the R103A/R115A double mutant.

Pressure adaptation of 3-isopropylmalate dehydrogenase from an extremely piezophilic bacterium is attributed to a single amino acid substitution.

3-Isopropylmalate dehydrogenase (IPMDH) from the extreme piezophile Shewanella benthica (SbIPMDH) is more pressure-tolerant than that from the atmospheric pressure-adapted Shewanella oneidensis (SoIPMDH). To understand the molecular mechanisms of this pressure tolerance, we analyzed mutated enzymes. The results indicate that only a single mutation at position 266, corresponding to Ala (SbIPMDH) and Ser (SoIPMDH), essentially affects activity under higher-pressure conditions.

High affinity nucleotide-binding mutant of the ε subunit of thermophilic F1-ATPase.

Specific ATP binding to the ε subunit of thermophilic F1-ATPase has been utilized for the biosensors of ATP in vivo. I report here that the ε subunit containing R103A/R115A mutations can bind ATP with a dissociation constant at 52 nM, which is two orders of magnitude higher affinity than the wild type. The mutant retained specificity for ATP; ADP and GTP bound to the mutant with dissociation constants 16 and 53 μM, respectively.

Severe MgADP inhibition of Bacillus subtilis F1-ATPase is not due to the absence of nucleotide binding to the noncatalytic nucleotide binding sites.

F1-ATPase from Bacillus subtilis (BF1) is severely suppressed by the MgADP inhibition. Here, we have tested if this is due to the loss of nucleotide binding to the noncatalytic site that is required for the activation. Measurements with a tryptophan mutant of BF1 indicated that the noncatalytic sites could bind ATP normally.

Pressure effects on the chimeric 3-isopropylmalate dehydrogenases of the deep-sea piezophilic Shewanella benthica and the atmospheric pressure-adapted Shewanella oneidensis.

The chimeric 3-isopropylmalate dehydrogenase enzymes were constructed from the deep-sea piezophilic Shewanella benthica and the shallow water Shewanella oneidensis genes. The properties of the enzymatic activities under pressure conditions indicated that the central region, which contained the active center and the dimer forming domains, was shown to be the most important region for pressure tolerance in the deep-sea enzyme.

ε subunit of Bacillus subtilis F1-ATPase relieves MgADP inhibition.

MgADP inhibition, which is considered as a part of the regulatory system of ATP synthase, is a well-known process common to all F1-ATPases, a soluble component of ATP synthase. The entrapment of inhibitory MgADP at catalytic sites terminates catalysis. Regulation by the ε subunit is a common mechanism among F1-ATPases from bacteria and plants.

ATP binding to the ϵ subunit of thermophilic ATP synthase is crucial for efficient coupling of ATPase and H+ pump activities.

ATP binding to the ϵ subunit of F1-ATPase, a soluble subcomplex of TFoF1 (FoF1-ATPase synthase from the thermophilic Bacillus strain PS3), affects the regulation of F1-ATPase activity by stabilizing the compact, ATPase-active, form of the ϵ subunit [Kato, S., Yoshida, M. and Kato-Yamada, Y.

Inhibition of thermophilic F-ATPase by the ε subunit takes different path from the ADP-Mg inhibition.

The F-ATPase, the soluble part of FoF-ATP synthase, is a rotary molecular motor consisting of αβγδε. The γ and ε subunits rotate relative to the αβδ sub-complex on ATP hydrolysis by the β subunit. The ε subunit is known as an endogenous inhibitor of the ATPase activity of the F-ATPase and is believed to function as a regulator of the ATP synthase.

Conformational transitions of subunit epsilon in ATP synthase from thermophilic Bacillus PS3.

Subunit epsilon of bacterial and chloroplast F(O)F(1)-ATP synthase is responsible for inhibition of ATPase activity. In Bacillus PS3 enzyme, subunit epsilon can adopt two conformations. In the "extended", inhibitory conformation, its two C-terminal alpha-helices are stretched along subunit gamma.

Modulation of nucleotide binding to the catalytic sites of thermophilic F(1)-ATPase by the epsilon subunit: implication for the role of the epsilon subunit in ATP synthesis.

Effect of epsilon subunit on the nucleotide binding to the catalytic sites of F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)) has been tested by using alpha(3)beta(3)gamma and alpha(3)beta(3)gammaepsilon complexes of TF(1) containing betaTyr341 to Trp substitution. The nucleotide binding was assessed with fluorescence quenching of the introduced Trp. The presence of the epsilon subunit weakened ADP binding to each catalytic site, especially to the highest affinity site.

Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators.

Adenosine 5'-triphosphate (ATP) is the major energy currency of cells and is involved in many cellular processes. However, there is no method for real-time monitoring of ATP levels inside individual living cells. To visualize ATP levels, we generated a series of fluorescence resonance energy transfer (FRET)-based indicators for ATP that were composed of the epsilon subunit of the bacterial F(o)F(1)-ATP synthase sandwiched by the cyan- and yellow-fluorescent proteins.

Role of the epsilon subunit of thermophilic F1-ATPase as a sensor for ATP.

The epsilon subunit of F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)) has been shown to bind ATP. The precise nature of the regulatory role of ATP binding to the epsilon subunit remains to be determined. To address this question, 11 mutants of the epsilon subunit were prepared, in which one of the basic or acidic residues was substituted with alanine.

Structures of the thermophilic F1-ATPase epsilon subunit suggesting ATP-regulated arm motion of its C-terminal domain in F1.

The epsilon subunit of bacterial and chloroplast F(o)F(1)-ATP synthases modulates their ATP hydrolysis activity. Here, we report the crystal structure of the ATP-bound epsilon subunit from a thermophilic Bacillus PS3 at 1.9-A resolution.

gammaepsilon Sub-complex of thermophilic ATP synthase has the ability to bind ATP.

The isolated epsilon subunit of F(1)-ATPase from thermophilic Bacillus PS3 (TF(1)) binds ATP [Y. Kato-Yamada, M. Yoshida, J.

Isolated epsilon subunit of Bacillus subtilis F1-ATPase binds ATP.

Previously, we demonstrated ATP binding to the isolated epsilon subunit of F1-ATPase from thermophilic Bacillus PS3 [Kato-Yamada Y., Yoshida M. (2003) J.

Real-time monitoring of conformational dynamics of the epsilon subunit in F1-ATPase.

It has been proposed that C-terminal two alpha-helices of the epsilon subunit of F1-ATPase can undergo conformational transition between retracted folded-hairpin form and extended form. Here, using F(1) from thermophilic Bacillus PS3, we monitored this transition in real time by fluorescence resonance energy transfer (FRET) between a donor dye and an acceptor dye attached to N terminus of the gamma subunit and C terminus of the epsilon subunit, respectively. High FRET (extended form) of F1 turned to low FRET (retracted form) by ATP, which then reverted as ATP was hydrolyzed to ADP.

Highly coupled ATP synthesis by F1-ATPase single molecules.

F1-ATPase is the smallest known rotary motor, and it rotates in an anticlockwise direction as it hydrolyses ATP. Single-molecule experiments point towards three catalytic events per turn, in agreement with the molecular structure of the complex. The physiological function of F1 is ATP synthesis.