Coli Enzyme Research Articles

Fructose-1-kinase has pleiotropic roles in Escherichia coli

In Escherichia coli, the master transcription regulator Catabolite Repressor Activator (Cra) regulates >100 genes in central metabolism. Cra binding to DNA is allosterically regulated by binding to fructose-1-phosphate (F-1-P), but the only documented source of F-1-P is from the concurrent import and phosphorylation of exogenous fructose. Thus, many have proposed that fructose-1,6-bisphosphate (F-1,6-BP) is also a physiological regulatory ligand. However, the role of F-1,6-BP has been widely debated. Here, we report that the E. coli enzyme fructose-1-kinase (FruK) can carry out its “reverse” reaction under physiological substrate concentrations to generate F-1-P from F-1,6-BP. We further show that FruK directly binds Cra with nanomolar affinity and forms higher order, heterocomplexes. Growth assays with a ΔfruK strain and fruK complementation show that FruK has a broader role in metabolism than fructose catabolism. Since fruK itself is repressed by Cra, these newly-reported events add layers to the dynamic regulation of E. coli central metabolism that occur in response to changing nutrients. These findings might have wide-spread relevance to other γ-proteobacteria, which conserve both Cra and FruK.

Role of Noradrenaline and Adrenoreceptors in Regulating Prostaglandin E2 Synthesis Cascade in Inflamed Endometrium of Pigs.

In the inflamed uterus, the production and secretion of prostaglandins (PGs) and noradrenergic innervation pattern are changed. Receptor-based control of prostaglandin E2 (PGE2) production and secretion by noradrenaline during uterine inflammation is unknown. The aim of this study was to determine the role of α1-, α2- and β-adrenoreceptors (ARs) in noradrenaline-influenced PG-endoperoxidase synthase-2 (PTGS-2) and microsomal PTGE synthase-1 (mPTGES-1) protein levels in the inflamed pig endometrium, and in the secretion of PGE2 from this tissue. E. coli suspension (E. coli group) or saline (CON group) was injected into the uterine horns. Eight days later, severe acute endometritis developed in the E. coli group. Endometrial explants were incubated with noradrenaline and/or α1-, α2- and β-AR antagonists. In the CON group, noradrenaline did not significantly change PTGS-2 and mPTGES-1 protein expression and increased PGE2 secretion compared to the control values (untreated tissue). In the E. coli group, both enzyme expression and PGE2 release were stimulated by noradrenaline, and these values were higher versus the CON group. The antagonists of α1- and α2-AR isoforms and β-AR subtypes do not significantly alter the noradrenaline effect on PTGS-2 and mPTGES-1 protein levels in the CON group, compared to noradrenaline action alone. In this group, α1A-, α2B- and β2-AR antagonists partly eliminated noradrenaline-stimulated PGE2 release. Compared to the noradrenaline effect alone, α1A-, α1B-, α2A-, α2B-, β1-, β2- and β3-AR antagonists together with noradrenaline reduced PTGS-2 protein expression in the E. coli group. Such effects were also exerted in this group by α1A-, α1D-, α2A-, β2- and β3-AR antagonists with noradrenaline on mPTGES-1 protein levels. In the E. coli group, the antagonists of all isoforms of α1-ARs and subtypes of β-ARs as well as α2A-ARs together with noradrenaline decreased PGE2 secretion versus noradrenaline action alone. Summarizing, in the inflamed pig endometrium, α1(A, B)-, α2(A, B)- and β(1, 2, 3)-ARs mediate the noradrenaline stimulatory effect on PTGE-2 protein expression, while noradrenaline via α1(A, D)-, α2A- and β(2, 3)-ARs increases mPTGES-1 protein expression and α1(A, B, D)-, α2A- and β(1, 2, 3)-ARs are involved in PGE2 release. Data suggest that noradrenaline may indirectly affect the processes regulated by PGE2 by influencing its production. Pharmacological modulation of particular AR isoforms/subtypes can be used to change PGE2 synthesis/secretion to alleviate inflammation and improve uterine function.

Mutating the environment of heme b595 of E. coli cytochrome bd-I oxidase shifts its redox potential by 200 mV without inactivating the enzyme

Cytochrome bd-I catalyzes the reduction of oxygen to water with the aid of hemes b558, b595 and d. Here, effects of a mutation of E445, a ligand of heme b595 and of R448, hydrogen bonded to E445 are studied electrochemically in the E. coli enzyme. The equilibrium potential of the three hemes are shifted by up to 200 mV in these mutants. Strikingly the E445D and the R448N mutants show a turnover of 41 ± 2 % and 20 ± 4 %, respectively. Electrocatalytic studies confirm that the mutants react with oxygen and bind and release NO. These results point towards the ability of cytochrome bd to react even if the electron transfer is less favorable.

Analysis of compound heterozygous and homozygous mutations found in peripheral subunits of human respiratory Complex I, NDUFS1, NDUFS2, NDUFS8 and NDUFV1, by modeling in the E. coli enzyme

Respiratory Complex I (NADH:ubiquinone oxidoreductase) is composed of 45 subunits, seven mitochondrially-encoded and 38 imported. Mutations in the nuclearly-encoded subunits have been regularly discovered in humans in recent years, and many lead to cardiomyopathy, Leigh Syndrome, and early death. From the literature, we have identified mutations at 17 different sites and constructed 31 mutants in a bacterial model system. Many of these mutations, found in NDUFS1, NDUFS2, NDUFS8, and NDUFV1, map to subunit interfaces, and we hypothesized that they would disrupt assembly of Complex I. The mutations were constructed in the homologous E. coli genes, nuoG, nuoCD, nuoI and nuoF, respectively, and expressed from a plasmid containing all Complex I genes. Membrane vesicles were prepared and rates of deamino-NADH oxidase activity measured, which indicated a range of reduced activity. Some mutants were also analyzed using recently developed assays of assembly, time-delayed expression, and co-immunoprecipitation, which showed that assembly was disrupted. With compound heterozygotes, we determined which mutation was more deleterious. Construction of alanine mutations allowed us to distinguish between phenotypes that were caused by loss of the original amino acid or introduction of the mutant residue.

Graphitic-C3N4/chitosan-doped NiO nanostructure to treat the polluted water and their bactericidal with in silico molecular docking analysis

Different concentrations (2 and 4 wt%) of graphite carbon nitride (g-C3N4) was doped into fixed amount of chitosan (CS) and nickel oxide (NiO) nanoparticles (NPs) via a co-precipitation route. The aim of study is to remove the pollutants from wastewater through catalytic activity (CA) and determine the bactericidal activities of synthesized products. X-ray diffraction pattern confirmed the cubic structure of NiO NPs and peak shifted to higher angle upon g-C3N4 doping. Fourier transform infrared spectroscopy revealed the existence of bending and stretching vibration mode. The absorption decreased gradually accompanied blue-shift and assessed bandgap energy increased upon doping. The high resolution transmission electron microscopy micrographs confirmed the formation of cubic-shaped NPs and elongated nanorods were seen for NiO and co-doped NiO. The catalytic efficiency of samples was examined using methylene blue (MB) in the presence of reducing agent. A remarkable dye de-colorization was confirmed with a g-C3N4 and CS doping; moreover, the bactericidal efficacy compared to Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was observed as 3.15 and 13.8 mm, respectively. In silico, molecular docking investigations targeting against b-lactamaseS. aureus and FabHE. coli enzymes assisted to elaborate the mechanism underlying microbicidal action of the NPs.

Optimisation of Benzodiazepine Immunoassay Using <i>β</i>-Glucuronidase Enzymatic Hydrolysis: A Comparison of Five Different <i>β</i>-Glucuronidase Enzymes

Background: Hydrolysis improves the sensitivity of drug detection for drug classes such as opiates/opioids and benzodiazepines, which are highly metabolized by glucuronidation and sulfation and should be implemented in analytical procedures to convert conjugated metabolites into the free or unbound form. This study was aimed to compare different enzymes to make an informed decision. Methods: In this study, the CEDIA Benzodiazepine assay was compared with the LC-MS-MS method using 150 positive urine samples and 50 negative urine samples. The samples were analysed without adding any enzyme and then by adding different enzymes to compare their performance. Results: The Kura Escherichia coli enzyme performed better than the Roche Escherichia coli enzyme which had 20% false-positive results. Kura BG-100 enzyme performed well but Kura B-One enzyme performed better The Kura B-One enzyme had only 11.5% false-positive results. When double the volume of Kura B-One enzyme was used to test to see if it will have any impact on reducing the number of false negatives, it performed worse. Kura Turbo enzyme behaved similarly to Kura BG-100. Conclusions: The β-glucuronidase enzymes comparison allowed us to identify the Kura B-One enzyme as the enzyme of choice for our operation because it reduces the false positives from 20% to 11.5% when compared with the Roche enzyme. It also improved the detection of oxazepam. The Kura B-One enzyme has a short incubation time for hydrolysis when used with the LC-MS-MS method. As a result, we improved the overall turn-around time and reduced the number of false positives that needed confirmation.

Inhibitory Compounds Targeting Plasmodium falciparum Gyrase B.

ABSTRACTMalaria persists as a major health problem due to the spread of drug resistance and the lack of effective vaccines. DNA gyrase is a well-validated and extremely effective therapeutic target in bacteria, and it is also known to be present in the apicoplast of malarial species, including Plasmodium falciparum. This raises the possibility that it could be a useful target for novel antimalarials. To date, characterization and screening of this gyrase have been hampered by difficulties in cloning and purification of the GyrA subunit, which is necessary together with GyrB for reconstitution of the holoenzyme. To overcome this, we employed a library of compounds with specificity for P. falciparum GyrB and assessed them in activity tests utilizing P. falciparum GyrB together with Escherichia coli GyrA to reconstitute a functional hybrid enzyme. Two inhibitory compounds were identified that preferentially inhibited the supercoiling activity of the hybrid enzyme over the E. coli enzyme. Of these, purpurogallin (PPG) was found to disrupt DNA binding to the hybrid gyrase complex and thus reduce the DNA-induced ATP hydrolysis of the enzyme. Binding studies indicated that PPG showed higher-affinity binding to P. falciparum GyrB than to the E. coli protein. We suggest that PPG achieves its inhibitory effect on gyrase through interaction with P. falciparum GyrB leading to disruption of DNA binding and, consequently, reduction of DNA-induced ATPase activity. The compound also showed an inhibitory effect against the malaria parasite in vitro and may be of interest for further development as an antimalarial agent.

Rimklb mutation causes male infertility in mice

Rimklb is a mammalian homologue of the E. coli enzyme RimK, which catalyzes addition of glutamic acid to the ribosomal protein S6. To date, no previous studies have shown any physiological role for Rimklb in mammals. In this study, using Western blotting, we found that Rimklb is distributed and expressed in mouse testis and heart. Rimklb was subsequently localized to the testicular Leydig cells using immunohistochemistry with an anti-Rimklb antibody. We generated a Rimklb mutant mouse in which a three-base deletion results in deletion of Ala 29 and substitution of Leu 30 with Val, which we named the RimklbA29del, L30V mutant mouse. RimklbA29del, L30V mutant mice show a decrease in testicular size and weight, and in vitro fertilization demonstrates complete male infertility. Furthermore, we found that a key factor in the mammalian target of the rapamycin/ribosomal protein S6 transcriptional pathway is hyperphosphorylated in the seminiferous tubules of the mutant testis. We conclude that Rimklb has important roles that include spermatogenesis in seminiferous tubules. In summary, male RimklbA29del, L30V mice are infertile.

Multi-omics Analysis of CRISPRi-Knockdowns Identifies Mechanisms that Buffer Decreases of Enzymes in E.coli Metabolism.

Enzymes maintain metabolism, and their concentration affects cellular fitness: high enzyme levels are costly, and low enzyme levels can limit metabolic flux. Here, we used CRISPR interference (CRISPRi) to study the consequences of decreasing E.coli enzymes below wild-type levels. A pooled CRISPRi screen with 7,177 strains demonstrates that metabolism buffers fitness defects for hours after the induction of CRISPRi. We characterized the metabolome and proteome responses in 30 CRISPRi strains and elucidated three gene-specific buffering mechanisms: ornithine buffered the knockdown of carbamoyl phosphate synthetase (CarAB) by increasing CarAB activity, S-adenosylmethionine buffered the knockdown of homocysteine transmethylase (MetE) by de-repressing expression of the methionine pathway, and 6-phosphogluconate buffered the knockdown of 6-phosphogluconate dehydrogenase (Gnd) by activating a bypass. In total, this work demonstrates that CRISPRi screens can reveal global sources of metabolic robustness and identify local regulatory mechanisms that buffer decreases of specific enzymes. A record of this paper's transparent peer review process is included in the Supplemental Information.

A ribonucleotide reductase from Clostridium botulinum reveals distinct evolutionary pathways to regulation via the overall activity site

Ribonucleotide reductase (RNR) is a central enzyme for the synthesis of DNA building blocks. Most aerobic organisms, including nearly all eukaryotes, have class I RNRs consisting of R1 and R2 subunits. The catalytic R1 subunit contains an overall activity site that can allosterically turn the enzyme on or off by the binding of ATP or dATP, respectively. The mechanism behind the ability to turn the enzyme off via the R1 subunit involves the formation of different types of R1 oligomers in most studied species and R1-R2 octamers in Escherichia coli To better understand the distribution of different oligomerization mechanisms, we characterized the enzyme from Clostridium botulinum, which belongs to a subclass of class I RNRs not studied before. The recombinantly expressed enzyme was analyzed by size-exclusion chromatography, gas-phase electrophoretic mobility macromolecular analysis, EM, X-ray crystallography, and enzyme assays. Interestingly, it shares the ability of the E. coli RNR to form inhibited R1-R2 octamers in the presence of dATP but, unlike the E. coli enzyme, cannot be turned off by combinations of ATP and dGTP/dTTP. A phylogenetic analysis of class I RNRs suggests that activity regulation is not ancestral but was gained after the first subclasses diverged and that RNR subclasses with inhibition mechanisms involving R1 oligomerization belong to a clade separated from the two subclasses forming R1-R2 octamers. These results give further insight into activity regulation in class I RNRs as an evolutionarily dynamic process.

DNA supercoiling differences in bacteria result from disparate DNA gyrase activation by polyamines.

DNA supercoiling is essential for all living cells because it controls all processes involving DNA. In bacteria, global DNA supercoiling results from the opposing activities of topoisomerase I, which relaxes DNA, and DNA gyrase, which compacts DNA. These enzymes are widely conserved, sharing >91% amino acid identity between the closely related species Escherichia coli and Salmonella enterica serovar Typhimurium. Why, then, do E. coli and Salmonella exhibit different DNA supercoiling when experiencing the same conditions? We now report that this surprising difference reflects disparate activation of their DNA gyrases by the polyamine spermidine and its precursor putrescine. In vitro, Salmonella DNA gyrase activity was sensitive to changes in putrescine concentration within the physiological range, whereas activity of the E. coli enzyme was not. In vivo, putrescine activated the Salmonella DNA gyrase and spermidine the E. coli enzyme. High extracellular Mg2+ decreased DNA supercoiling exclusively in Salmonella by reducing the putrescine concentration. Our results establish the basis for the differences in global DNA supercoiling between E. coli and Salmonella, define a signal transduction pathway regulating DNA supercoiling, and identify potential targets for antibacterial agents.

A Proposed Proton Relay Mechanism in Methylenetetrahydrofolate Reductase

Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10‐methylenetetrahydrofolate (CH2‐H4folate) to 5‐methyltetrahydrofolate (CH3‐H4folate) by NAD(P)H. This reaction can be divided into two half‐reactions: the NAD(P)H reductive halfreaction and the folate oxidative half‐reaction. The X‐ray crystal structure of the E. coli enzyme has revealed a hydrogen‐bonded network of three active site amino acids, Glu28, Ser26 and His273. Previous studies with a Glu28Gln mutant indicated that Glu28, located near N10 of CH3‐H4folate, is an essential amino acid. Since Ser26 and His273 form hydrogen bonds to Glu28, we propose that Ser26, His273 and Glu28 are involved in a proton relay mechanism which donates a proton to CH2‐H4folate in the folate oxidative half‐reaction. In order to elucidate the role of Ser26 in this mechanism, we have characterized Ser26Thr and Ser26Ala mutants using the steady‐state CH3‐H4folate‐menadione oxidoreductase assay, in which the folate oxidative half‐reaction is rate limiting. We have hypothesized that the Ser26Thr mutant will have activity comparable to the wild‐type enzyme and the Ser26Ala mutant will have reduced activity due to its inability to hydrogen bond. Contrary to our hypothesis, we show that both mutants catalyze the folate reaction similar to the wild‐type enzyme. We also demonstrate that the Ser26Ala mutant shows competitive substrate inhibition in the CH3‐H4folate‐menadione assay, which is not observed for the wild‐type enzyme. Examination of the mutants in the NADH‐menadione assay shows kinetic parameters similar to those of the wild‐type enzyme. Taken together, our results suggest that Ser26 may not play an important role in either folate or NADH catalysis of MTHFR. However, we propose that in the case of the folate reaction, a water molecule inserted between the alanine and His273 might function like a serine in the proton relay mechanism, explaining the lack of a defect in the Ser26Ala mutant. To investigate the proton relay mechanism and the role of Ser26 further, we are studying the pH‐dependency of MTHFR in the CH3‐H4folate‐menadione assay.

Structures of the ATP-fueled ClpXP proteolytic machine bound to protein substrate.

ClpXP is an ATP-dependent protease in which the ClpX AAA+ motor binds, unfolds, and translocates specific protein substrates into the degradation chamber of ClpP. We present cryo-EM studies of the E. coli enzyme that show how asymmetric hexameric rings of ClpX bind symmetric heptameric rings of ClpP and interact with protein substrates. Subunits in the ClpX hexamer assume a spiral conformation and interact with two-residue segments of substrate in the axial channel, as observed for other AAA+ proteases and protein-remodeling machines. Strictly sequential models of ATP hydrolysis and a power stroke that moves two residues of the substrate per translocation step have been inferred from these structural features for other AAA+ unfoldases, but biochemical and single-molecule biophysical studies indicate that ClpXP operates by a probabilistic mechanism in which five to eight residues are translocated for each ATP hydrolyzed. We propose structure-based models that could account for the functional results.

Dinucleoside polyphosphates act as 5\u2032-RNA caps in bacteria

It has been more than 50 years since the discovery of dinucleoside polyphosphates (NpnNs) and yet their roles and mechanisms of action remain unclear. Here, we show that both methylated and non-methylated NpnNs serve as RNA caps in Escherichia coli. NpnNs are excellent substrates for T7 and E. coli RNA polymerases (RNAPs) and efficiently initiate transcription. We demonstrate, that the E. coli enzymes RNA 5′-pyrophosphohydrolase (RppH) and bis(5′-nucleosyl)-tetraphosphatase (ApaH) are able to remove the NpnN-caps from RNA. ApaH is able to cleave all NpnN-caps, while RppH is unable to cleave the methylated forms suggesting that the methylation adds an additional layer to RNA stability regulation. Our work introduces a different perspective on the chemical structure of RNA in prokaryotes and on the role of RNA caps. We bring evidence that small molecules, such as NpnNs are incorporated into RNA and may thus influence the cellular metabolism and RNA turnover.

Unique active site formation in a novel galactose 1-phosphate uridylyltransferase from the hyperthermophilic archaeon Pyrobaculum aerophilum.

A gene encoding galactose 1-phosphate uridylyltransferase (GalT) was identified in the hyperthermophilic archaeon Pyrobaculum aerophilum. The gene was overexpressed in Escherichia coli, after which its product was purified and characterized. The expressed enzyme was highly thermostable and retained about 90% of its activity after incubation for 10 minutes at temperatures up to 90°C. Two different crystal structures of P. aerophilum GalT were determined: the substrate-free enzyme at 2.33 Å and the UDP-bound H140F mutant enzyme at 1.78 Å. The main-chain coordinates of the P. aerophilum GalT monomer were similar to those in the structures of the E. coli and human GalTs, as was the dimeric arrangement. However, there was a striking topological difference between P. aerophilum GalT and the other two enzymes. In the E. coli and human enzymes, the N-terminal chain extends from one subunit into the other and forms part of the substrate-binding pocket in the neighboring subunit. By contrast, the N-terminal chain in P. aerophilum GalT extends to the substrate-binding site in the same subunit. Amino acid sequence alignment showed that a shorter surface loop in the N-terminal region contributes to the unique topology of P. aerophilum GalT. Structural comparison of the substrate-free enzyme with UDP-bound H140F suggests that binding of the glucose moiety of the substrate, but not the UDP moiety, gives rise to a large structural change around the active site. This may in turn provide an appropriate environment for the enzyme reaction.

Homologous bd oxidases share the same architecture but differ in mechanism

Cytochrome bd oxidases are terminal reductases of bacterial and archaeal respiratory chains. The enzyme couples the oxidation of ubiquinol or menaquinol with the reduction of dioxygen to water, thus contributing to the generation of the protonmotive force. Here, we determine the structure of the Escherichia coli bd oxidase treated with the specific inhibitor aurachin by cryo-electron microscopy (cryo-EM). The major subunits CydA and CydB are related by a pseudo two fold symmetry. The heme b and d cofactors are found in CydA, while ubiquinone-8 is bound at the homologous positions in CydB to stabilize its structure. The architecture of the E. coli enzyme is highly similar to that of Geobacillus thermodenitrificans, however, the positions of heme b595 and d are interchanged, and a common oxygen channel is blocked by a fourth subunit and substituted by a more narrow, alternative channel. Thus, with the same overall fold, the homologous enzymes exhibit a different mechanism.

Dynamic origins of substrate promiscuity in bacterial galactokinases

Galactokinase catalyses the ATP-dependent phosphorylation of galactose and structurally related sugars. The enzyme has attracted interest as a potential biocatalyst for the production of sugar 1-phosphates and several attempts have been made to broaden its specificity. In general, bacterial galactokinases have wider substrate ranges than mammalian ones. The enzymes from Escherichia coli and Lactococcus lactis have received particular attention and a number of variants with increased promiscuity have been identified. Here, we present a molecular dynamics study designed to investigate the molecular causes of the wider substrate ranges of these enzymes and their variants with particular reference to protein mobility. Some regions close to the active site of the enzyme have different structures in the bacterial enzymes compared to the human one. Alterations known to increase the substrate range (e.g. Y371H in the E. coli enzyme), tend to alter the conformation of a key α-helical region (residues 216–232 in the E. coli enzyme). The equivalent helix in the human enzyme has previously been predicted to be altered in variants which affect catalytic activity or protein stability. This helix appears to be a key region in galactokinases from a range of species and may represent an interesting target for future attempts to broaden the specificity of galactokinases.

Mapping of a Regulatory Site of the Escherichia coli ADP-Glucose Pyrophosphorylase.

The enzyme ADP-glucose pyrophosphorylase (ADP-Glc PPase) controls the biosynthesis of glycogen in bacteria and starch in plants. It is regulated by various activators in different organisms according to their metabolic characteristics. In Escherichia coli, the major allosteric activator is fructose 1,6-bisphosphate (FBP). Other potent activator analogs include 1,6-hexanediol bisphosphate (HBP) and pyridoxal 5′-phosphate (PLP). Recently, a crystal structure with FBP bound was reported (PDB ID: 5L6S). However, it is possible that the FBP site found is not directly responsible for the activation of the enzyme. We hypothesized FBP activates by binding one of its phosphate groups to another site (“P1”) in which a sulfate molecule was observed. In the E. coli enzyme, Arg40, Arg52, and Arg386 are part of this “P1” pocket and tightly complex this sulfate, which is also present in the crystal structures of ADP-Glc PPases from Agrobacterium tumefaciens and Solanum tuberosum. To test this hypothesis, we modeled alternative binding conformations of FBP, HBP, and PLP into “P1.” In addition, we performed a scanning mutagenesis of Arg residues near potential phosphate binding sites (“P1,” “P2,” “P3”). We found that Arg40 and Arg52 are essential for FBP and PLP binding and activation. In addition, mutation of Arg386 to Ala decreased the apparent affinity for the activators more than 35-fold. We propose that the activator binds at this “P1” pocket, as well as “P2.” Arg40 and Arg52 are highly conserved residues and they may be a common feature to complex the phosphate moiety of different sugar phosphate activators in the ADP-Glc PPase family.

Unlike the Escherichia coli counterpart, archaeal RNase HII cannot process ribose monophosphate abasic sites and oxidized ribonucleotides embedded in DNA

The presence of ribonucleoside monophosphates (rNMPs) in nuclear DNA decreases genome stability. To ensure survival despite rNMP insertions, cells have evolved a complex network of DNA repair mechanisms, in which the ribonucleotide excision repair pathway, initiated by type 2 RNase H (RNase HII/2), plays a major role. We recently demonstrated that eukaryotic RNase H2 cannot repair damage, that is, ribose monophosphate abasic (both apurinic or apyrimidinic) site (rAP) or oxidized rNMP embedded in DNA. Currently, it remains unclear why RNase H2 is unable to repair these modified nucleic acids having either only a sugar moiety or an oxidized base. Here, we compared the endoribonuclease specificity of the RNase HII enzymes from the archaeon Pyrococcus abyssi and the bacterium Escherichia coli, examining their ability to process damaged rNMPs embedded in DNA in vitro We found that E. coli RNase HII cleaves both rAP and oxidized rNMP sites. In contrast, like the eukaryotic RNase H2, P. abyssi RNase HII did not display any rAP or oxidized rNMP incision activities, even though it recognized them. Notably, the archaeal enzyme was also inactive on a mismatched rNMP, whereas the E. coli enzyme displayed a strong preference for the mispaired rNMP over the paired rNMP in DNA. On the basis of our biochemical findings and also structural modeling analyses of RNase HII/2 proteins from organisms belonging to all three domains of life, we propose that RNases HII/2's dual roles in ribonucleotide excision repair and RNA/DNA hydrolysis result in limited acceptance of modified rNMPs embedded in DNA.

The proton pumping bo oxidase from Vitreoscilla

The cytochrome bo3 quinol oxidase from Vitreoscilla (vbo3) catalyses oxidation of ubiquinol and reduction of O2 to H2O. Data from earlier studies suggested that the free energy released in this reaction is used to pump sodium ions instead of protons across a membrane. Here, we have studied the functional properties of heterologously expressed vbo3 with a variety of methods. (i) Following oxygen consumption with a Clark-type electrode, we did not observe a measurable effect of Na+ on the oxidase activity of purified vbo3 solubilized in detergent or reconstituted in liposomes. (ii) Using fluorescent dyes, we find that vbo3 does not pump Na+ ions, but H+ across the membrane, and that H+-pumping is not influenced by the presence of Na+. (iii) Using an oxygen pulse method, it was found that 2 H+/e− are ejected from proteoliposomes, in agreement with the values found for the H+-pumping bo3 oxidase of Escherichia coli (ecbo3). This coincides with the interpretation that 1 H+/e− is pumped across the membrane and 1 H+/e− is released during quinol oxidation. (iv) When the electron transfer kinetics of vbo3 upon reaction with oxygen were followed in single turnover experiments, a similar sequence of reaction steps was observed as reported for the E. coli enzyme and none of these reactions was notably affected by the presence of Na+. Overall the data show that vbo3 is a proton pumping terminal oxidase, behaving similarly to the Escherichia coli bo3 quinol oxidase.