The "life-span" of lytic polysaccharide monooxygenases (LPMOs) correlates to the number of turnovers in the reductant peroxidase reaction.

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that degrade the insoluble crystalline polysaccharides cellulose and chitin. Besides the H2O2 cosubstrate, the cleavage of glycosidic bonds by LPMOs depends on the presence of a reductant needed to bring the enzyme into its reduced, catalytically active Cu(I) state. Reduced LPMOs that are not bound to substrate catalyze reductant peroxidase reactions, which may lead to oxidative damage and irreversible inactivation of the enzyme. However, the kinetics of this reaction remain largely unknown, as do possible variations between LPMOs belonging to different families. Here, we describe the kinetic characterization of two fungal family AA9 LPMOs, TrAA9A of Trichoderma reesei and NcAA9C of Neurospora crassa, and two bacterial AA10 LPMOs, ScAA10C of Streptomyces coelicolor and SmAA10A of Serratia marcescens. We found peroxidation of ascorbic acid and methyl-hydroquinone resulted in the same probability of LPMO inactivation (pi), suggesting that inactivation is independent of the nature of the reductant. We showed the fungal enzymes were clearly more resistant toward inactivation, having pi values of less than 0.01, whereas the pi for SmAA10A was an order of magnitude higher. However, the fungal enzymes also showed higher catalytic efficiencies (kcat/KM(H2O2)) for the reductant peroxidase reaction. This inverse linear correlation between the kcat/KM(H2O2) and pi suggests that, although having different life spans in terms of the number of turnovers in the reductant peroxidase reaction, LPMOs that are not bound to substrates have similar half-lives. These findings have not only potential biological but also industrial implications.

Dietary-derived vitamin B12 protects Caenorhabditis elegans from thiol-reducing agents.

BACKGROUND: One-carbon metabolism, which includes the folate and methionine cycles, involves the transfer of methyl groups which are then utilised as a part of multiple physiological processes including redox defence. During the methionine cycle, the vitamin B12-dependent enzyme methionine synthetase converts homocysteine to methionine. The enzyme S-adenosylmethionine (SAM) synthetase then uses methionine in the production of the reactive methyl carrier SAM. SAM-binding methyltransferases then utilise SAM as a cofactor to methylate proteins, small molecules, lipids, and nucleic acids. RESULTS: We describe a novel SAM methyltransferase, RIPS-1, which was the single gene identified from forward genetic screens in Caenorhabditis elegans looking for resistance to lethal concentrations of the thiol-reducing agent dithiothreitol (DTT). As well as RIPS-1 mutation, we show that in wild-type worms, DTT toxicity can be overcome by modulating vitamin B12 levels, either by using growth media and/or bacterial food that provide higher levels of vitamin B12 or by vitamin B12 supplementation. We show that active methionine synthetase is required for vitamin B12-mediated DTT resistance in wild types but is not required for resistance resulting from RIPS-1 mutation and that susceptibility to DTT is partially suppressed by methionine supplementation. A targeted RNAi modifier screen identified the mitochondrial enzyme methylmalonyl-CoA epimerase as a strong genetic enhancer of DTT resistance in a RIPS-1 mutant. We show that RIPS-1 is expressed in the intestinal and hypodermal tissues of the nematode and that treating with DTT, ß-mercaptoethanol, or hydrogen sulfide induces RIPS-1 expression. We demonstrate that RIPS-1 expression is controlled by the hypoxia-inducible factor pathway and that homologues of RIPS-1 are found in a small subset of eukaryotes and bacteria, many of which can adapt to fluctuations in environmental oxygen levels. CONCLUSIONS: This work highlights the central importance of dietary vitamin B12 in normal metabolic processes in C. elegans, defines a new role for this vitamin in countering reductive stress, and identifies RIPS-1 as a novel methyltransferase in the methionine cycle.

A Reduced F420-Dependent Nitrite Reductase in an Anaerobic Methanotrophic Archaeon.

Anaerobic methanotrophic archaea (ANME), which oxidize methane in marine sediments through syntrophic associations with sulfate-reducing bacteria, carry homologs of coenzyme F420-dependent sulfite reductase (Fsr) of Methanocaldococcus jannaschii, a hyperthermophilic methanogen from deep-sea hydrothermal vents. M. jannaschii Fsr (MjFsr) and ANME-Fsr belong to two phylogenetically distinct groups, FsrI and FsrII, respectively. MjFsrI reduces sulfite to sulfide with reduced F420 (F420H2), protecting methyl coenzyme M reductase (Mcr), an essential enzyme for methanogens, from sulfite inhibition. However, the function of FsrIIs in ANME, which also rely on Mcr and live in sulfidic environments, is unknown. We have determined the catalytic properties of FsrII from a member of ANME-2c. Since ANME remain to be isolated, we expressed ANME2c-FsrII in a closely related methanogen, Methanosarcina acetivorans. Purified recombinant FsrII contained siroheme, indicating that the methanogen, which lacks a native sulfite reductase, produced this coenzyme. Unexpectedly, FsrII could not reduce sulfite or thiosulfate with F420H2. Instead, it acted as an F420H2-dependent nitrite reductase (FNiR) with physiologically relevant Km values (nitrite, 5 µM; F420H2, 14 µM). From kinetic, thermodynamic, and structural analyses, we hypothesize that in FNiR, F420H2-derived electrons are delivered at the oxyanion reduction site at a redox potential that is suitable for reducing nitrite (E0' [standard potential], +440 mV) but not sulfite (E0', -116 mV). These findings and the known nitrite sensitivity of Mcr suggest that FNiR may protect nondenitrifying ANME from nitrite toxicity. Remarkably, by reorganizing the reductant processing system, Fsr transforms two analogous oxyanions in two distinct archaeal lineages with different physiologies and ecologies. IMPORTANCE Coenzyme F420-dependent sulfite reductase (Fsr) protects methanogenic archaea inhabiting deep-sea hydrothermal vents from the inactivation of methyl coenzyme M reductase (Mcr), one of their essential energy production enzymes. Anaerobic methanotrophic archaea (ANME) that oxidize methane and rely on Mcr, carry Fsr homologs that form a distinct clade. We show that a member of this clade from ANME-2c functions as F420-dependent nitrite reductase (FNiR) and lacks Fsr activity. This specialization arose from a distinct feature of the reductant processing system and not the substrate recognition element. We hypothesize FNiR may protect ANME Mcr from inactivation by nitrite. This is an example of functional specialization within a protein family that is induced by changes in electron transfer modules to fit an ecological need.

Reprint of: Ubisemiquinone Is the Electron Donor for Superoxide Formation by Complex III of Heart Mitochondria.

Much evidence indicates that superoxide is generated from O2 in a cyanide-sensitive reaction involving a reduced component of complex III of the mitochondrial respiratory chain, particularly when antimycin A is present. Although it is generally believed that ubisemiquinone is the electron donor to O2, little experimental evidence supporting this view has been reported. Experiments with succinate as electron donor in the presence of antimycin A in intact rat heart mitochondria, which contain much superoxide dismutase but little catalase, showed that myxothiazol, which inhibits reduction of the Rieske iron-sulfur center, prevented formation of hydrogen peroxide, determined spectrophotometrically as the H2O2-peroxidase complex. Similarly, depletion of the mitochondria of their cytochrome c also inhibited formation of H2O2, which was restored by addition of cytochrome c. These observations indicate that factors preventing the formation of ubisemiquinone also prevent H2O2 formation. They also exclude ubiquinol, which remains reduced under these conditions, as the reductant of O2. Since cytochrome b also remains fully reduced when myxothiazol is added to succinate- and antimycin A-supplemented mitochondria, reduced cytochrome b may also be excluded as the reductant of O2. These observations, which are consistent with the Q-cycle reactions, by exclusion of other possibilities leave ubisemiquinone as the only reduced electron carrier in complex III capable of reducing O2 to O2-.

pH dependence of photosystem II photoinhibition: relationship with structural transition of oxygen-evolving complex at the pH of thylakoid lumen.

Ca-depleted photosystem II membranes (PSII[-Ca]) do not contain PsbP and PsbQ proteins protecting the Mn4CaO5 cluster of the PSII oxygen-evolving complex (OEC). Therefore, the Mn ions in the PSII(-Ca) membranes can be reduced by exogenous bulky reductants or the charged reductant Fe(II). We have recently found that the resistance of Mn ions in the OEC to the Fe(II) action is pH dependent and that this reductant is less effective at pH 5.7 than at pH 6.5 (Semin et al. J Photochem Photobiol B 178:192, 2018). Taking these data into account, we investigated the photoinhibition in different PSII membranes at pH 5.7 and 6.5 and found that the resistance to photoinhibition of PSII and PSII(-Ca) membranes with a Mn cluster is higher at pH 5.7 than at pH 6.5, whereas the resistance of the Mn-depleted PSII membranes is pH independent. In thylakoids, light generates the transmembrane ΔpH, leading to the acidulation of lumen that results in pH 5.7. The uncouplers (NH4Cl or nigericin) that significantly prevent acidulation increase the rate of PSII photoinhibition in thylakoids. We suggest that the structural transition in the OEC at pH 5.7 plays a role of a built-in mechanism increasing the resistance of OEC to photoinhibition under illumination, since it is accompanied by a pH decrease in lumen to 5.7. The coincidence of these pH values, i.e. lumen pH under illumination and pH of the maximal resistance of the Mn cluster to the reduction by reductants, can point at the pH-dependent mechanism of PSII self-protection from photoinactivation.

Hydrogen Does Not Appear To Be a Major Electron Donor for Symbiosis with the Deep-Sea Hydrothermal Vent Tubeworm Riftia pachyptila.

Use of hydrogen gas (H2) as an electron donor is common among free-living chemolithotrophic microorganisms. Given the presence of this dissolved gas at deep-sea hydrothermal vents, it has been suggested that it may also be a major electron donor for the free-living and symbiotic chemolithoautotrophic bacteria that are the primary producers at these sites. Giant Riftia pachyptila siboglinid tubeworms and their symbiotic bacteria ("Candidatus Endoriftia persephone") dominate many vents in the Eastern Pacific, and their use of sulfide as a major electron donor has been documented. Genes encoding hydrogenase are present in the "Ca Endoriftia persephone" genome, and proteome data suggest that these genes are expressed. In this study, high-pressure respirometry of intact R. pachyptila and incubations of trophosome homogenate were used to determine whether this symbiotic association could also use H2 as a major electron donor. Measured rates of H2 uptake by intact R. pachyptila in high-pressure respirometers were similar to rates measured in the absence of tubeworms. Oxygen uptake rates in the presence of H2 were always markedly lower than those measured in the presence of sulfide, as was the incorporation of 13C-labeled dissolved inorganic carbon. Carbon fixation by trophosome homogenate was not stimulated by H2, nor was hydrogenase activity detectable in these samples. Though genes encoding [NiFe] group 1e and [NiFe] group 3b hydrogenases are present in the genome and transcribed, it does not appear that H2 is a major electron donor for this system, and it may instead play a role in intracellular redox homeostasis.IMPORTANCE Despite the presence of hydrogenase genes, transcripts, and proteins in the "Ca Endoriftia persephone" genome, transcriptome, and proteome, it does not appear that R. pachyptila can use H2 as a major electron donor. For many uncultivable microorganisms, omic analyses are the basis for inferences about their activities in situ However, as is apparent from the study reported here, there are dangers in extrapolating from omics data to function, and it is essential, whenever possible, to verify functions predicted from omics data with physiological and biochemical measurements.

Antitumor Platinium(IV) Prodrugs: A Systematic Computational Exploration of Their Reduction Mechanism by l-Ascorbic Acid.

The reduction mechanism of Pt(IV) anticancer prodrugs, still today a matter of debate, assisted by one of the dominant reductants in human plasma, that is l-ascorbic acid in its monodeprotonated form, has been computationally examined in this work. In order to check what should be the influence on the reduction rate of the identity of the ligands in axial and equatorial position, both cisplatin and oxaliplatin derivatives have been studied, varying the ligands in axial position in connection with the role they should play as bridges, trans leaving species, and proton acceptors. OH, OAc, Cl, and Br ligands have been tested as bridging/leaving ligands, whereas Cl and aspirin have been used as trans labile and less labile ligands, respectively. The most recent theoretical and experimental investigations have demonstrated that the generally adopted grouping of reduction mechanisms into inner- and outer-sphere does not properly take into account all the viable alternatives. Therefore, inner-sphere mechanisms, classified as ligand-bridged, ligand-bridged-H transfer and enolate ß-carbon attack, have been explored for all the complexes under investigation. Concerning the outer-sphere mechanism, redox potentials have been calculated adopting a recently proposed procedure based on the separation between electrochemical and chemical events to evaluate their propensity to be reduced. Moreover, according to the hypothesis that the outer-sphere reduction mechanism involves the sequential addition of two electrons causing the formation of a Pt(III) intermediate, the possibility that singlet and triplet pathways can cross for the Pt(IV) cisplatin derivative having two chlorido ligands in axial position has been explored in detail. Results show that the mechanism indicated as base-assisted outer sphere can become competitive with respect to the inner one if two singlet-triplet spin inversions occur. Results presented here are helpful in addressing synthetic strategies as they show that Pt(IV) prodrugs propensity to be reduced can be properly tuned and give indications on how this aim can be accomplished.

Investigations of the Kinetics and Mechanism of Reduction of a Carboplatin Pt(IV) Prodrug by the Major Small-Molecule Reductants in Human Plasma.

The development of Pt(IV) anticancer prodrugs to overcome the detrimental side effects of Pt(II)-based anticancer drugs is of current interest. The kinetics and reaction mechanisms of the reductive activation of the carboplatin Pt(IV) prodrug cis,trans-[Pt(cbdca)(NH3)2Cl2] (cbdca = cyclobutane-1,1-dicarboxylate) by the major small-molecule reductants in human plasma were analyzed in this work. The reductants included ascorbate (Asc), the thiol-containing molecules L-cysteine (Cys), DL-homocysteine (Hcy), and glutathione (GSH), and the dipeptide Cys-Gly. Overall second-order kinetics were established in all cases. At the physiological pH of 7.4, the observed second-order rate constants k' followed the order Asc << Cys-Gly ~ Hcy < GSH < Cys. This reactivity order together with the abundances of the reductants in human plasma indicated Cys as the major small-molecule reductant in vivo, followed by GSH and ascorbate, whereas Hcy is much less important. In the cases of Cys and GSH, detailed reaction mechanisms and the reactivity of the various protolytic species at physiological pH were derived. The rate constants of the rate-determining steps were evaluated, allowing the construction of reactivity-versus-pH distribution diagrams for Cys and GSH. The diagrams unraveled that species III of Cys (-SCH2CH(NH3+)COO-) and species IV of GSH (-OOCCH(NH3+)CH2CH2CONHCH(CH2S-)- CONHCH2COO-) were exclusively dominant in the reduction process. These two species are anticipated to be of pivotal importance in the reduction of other types of Pt(IV) prodrugs as well.

Unraveling the interactions of the physiological reductant flavodoxin with the different conformations of the Fe protein in the nitrogenase cycle.

Nitrogenase reduces dinitrogen (N2) to ammonia in biological nitrogen fixation. The nitrogenase Fe protein cycle involves a transient association between the reduced, MgATP-bound Fe protein and the MoFe protein and includes electron transfer, ATP hydrolysis, release of Pi, and dissociation of the oxidized, MgADP-bound Fe protein from the MoFe protein. The cycle is completed by reduction of oxidized Fe protein and nucleotide exchange. Recently, a kinetic study of the nitrogenase Fe protein cycle involving the physiological reductant flavodoxin reported a major revision of the rate-limiting step from MoFe protein and Fe protein dissociation to release of Pi Because the Fe protein cannot interact with flavodoxin and the MoFe protein simultaneously, knowledge of the interactions between flavodoxin and the different nucleotide states of the Fe protein is critically important for understanding the Fe protein cycle. Here we used time-resolved limited proteolysis and chemical cross-linking to examine nucleotide-induced structural changes in the Fe protein and their effects on interactions with flavodoxin. Differences in proteolytic cleavage patterns and chemical cross-linking patterns were consistent with known nucleotide-induced structural differences in the Fe protein and indicated that MgATP-bound Fe protein resembles the structure of the Fe protein in the stabilized nitrogenase complex structures. Docking models and cross-linking patterns between the Fe protein and flavodoxin revealed that the MgADP-bound state of the Fe protein has the most complementary docking interface with flavodoxin compared with the MgATP-bound state. Together, these findings provide new insights into the control mechanisms in protein-protein interactions during the Fe protein cycle.

Palladium nanoparticles synthesis, characterization using glucosamine as the reductant and stabilizing agent to explore their antibacterial & catalytic applications.

Low cost and an easy technique for the synthesis of palladium nanoparticles (PdNPs) was developed. Glucosamine was used to stabilize palladium precursor (PdCl2) into palladium nanoparticles. Several analytical techniques were used for the determination of morphology, crystalline structure; size, capping, and composition of synthesize palladium nanoparticles. The UV-visible spectroscopy SPR peak (Surface Plasmon Resonance) at 284 nm revealed synthesis of PdNPs. Energy dispersive X-ray (EDX) and X-ray diffraction (XRD) studies proved the elemental composition and crystalline structure of the synthesized palladium nanoparticles respectively. The average particle sizes (5.5 nm) were obtained by using the 1 M glucosamine solution, with a fixed amount of PdCl2 (4 mM). Moreover, the as synthesized PdNPs was evaluated against Gram negative bacterial E. which shows tremendous antibacterial activity as compare to tobramycin standard antibiotics. It's mechanistically found that PdNPs damage cell membrane and caused imbalance of metabolism system of the cell as a result production of reactive oxygen species (ROS). Thus, these finding revealed that cells become leaky and all organelles come out from cells, finally caused death of the E. coli. Addition, the as prepared PdNPs also showed excellent catalytic activities toward reduction of methylene blue and 4-nitrophenol.Thus, glucosamine mediated PdNPs having dual functions biomedical as well as intoxicating catalyst for industries.

Amino acids located in the outer-sphere of the trinuclear copper center in a multicopper oxidase, CueO as the putative electron donor in the four-electron reduction of dioxygen.

The reaction mechanism of multicopper oxidase (MCO) to reduce dioxygen to water has not been fully understood yet in spite of extensive studies including on the intermediate I (peroxide intermediate) and intermediate II (native intermediate with an O-centered structure at the trinuclear copper center (TNC)). We performed the Phe mutations at the four amino acids, Tyr69, Cys138, Trp139, and Tyr496 located in the outer-sphere of TNC in CueO at the aim of studying whether they play a role as the fourth electron donor to dioxygen or not. Spectral properties and enzymatic activities of CueO were sparingly affected or not affected by the mutations at these putative electron donors. Of the targeted four amino acids Trp139 is in a d-π interaction distance with one of T3Cus and drives stepwise formation and release of water molecules by making two T3Cus non-equivalent. However, contribution of a radical species derived from Trp139 has not been observed in the formation and decay processes of the reaction intermediates. The present study strongly suggests that the amino acids located in the outer-sphere of TNC are not utilized as electron donor in the reduction of dioxygen to water by the three-domain MCO, CueO, differing from cytochrome oxidase and SLAC, a two-domain MCO, in which reaction participation of an uncoordinated Tyr residue has been proposed. SUMMARY: We performed the Phe mutations at the four amino acids, Tyr69, Cys138, Trp139 and Tyr496 located in the outer-coordination sphere of the trinuclear copper center in a three-domain multicopper oxidase, CueO to ascertain whether they function as an electron donor or not in the four-electron reduction of dioxygen. Characterizations of the mutants and reactions did not suggest participation of the targeted amino acids, indicating that CueO follows a different reaction mechanism from that of a two-domain multicopper oxidase, SLAC, in which reaction participation of an uncoordinated Tyr has been suggested.

The class III ribonucleotide reductase from Neisseria bacilliformis can utilize thioredoxin as a reductant.

The class III anaerobic ribonucleotide reductases (RNRs) studied to date couple the reduction of ribonucleotides to deoxynucleotides with the oxidation of formate to CO2. Here we report the cloning and heterologous expression of the Neisseria bacilliformis class III RNR and show that it can catalyze nucleotide reduction using the ubiquitous thioredoxin/thioredoxin reductase/NADPH system. We present a structural model based on a crystal structure of the homologous Thermotoga maritima class III RNR, showing its architecture and the position of conserved residues in the active site. Phylogenetic studies suggest that this form of class III RNR is present in bacteria and archaea that carry out diverse types of anaerobic metabolism.

NAD+ as a Hydride Donor and Reductant.

Reduced nicotinamide adenine dinucleotide (NADH) can generate a ruthenium-hydride intermediate that catalyzes the reduction of O2 to H2O2, which endows it with potent anticancer properties. A catalyst that could access a Ru-H intermediate using oxidized nicotinamide adenine dinucleotide (NAD+) as the H- source, however, could draw upon a supply of reducing equivalents 1000-fold more abundant than NADH, which would enable significantly greater H2O2 production. Herein, it is demonstrated, using the reduction of ABTS•- to ABTS2-, that NAD+ can function as a reductant. Mechanistic evidence is presented that suggests a Ru-H intermediate is formed via ß-hydride elimination from a ribose subunit in NAD+. The insight gained from the heretofore unknown ability of NAD+ to function as a reductant and H- donor may lead to undiscovered biological carbohydrate oxidation pathways and new chemotherapeutic strategies.

Involutin is an Fe3+ reductant secreted by the ectomycorrhizal fungus Paxillus involutus during Fenton-based decomposition of organic matter.

Ectomycorrhizal fungi play a key role in mobilizing nutrients embedded in recalcitrant organic matter complexes, thereby increasing nutrient accessibility to the host plant. Recent studies have shown that during the assimilation of nutrients, the ectomycorrhizal fungus Paxillus involutus decomposes organic matter using an oxidative mechanism involving Fenton chemistry (Fe(2+) + H2O2 + H(+) → Fe(3+) + ˙OH + H2O), similar to that of brown rot wood-decaying fungi. In such fungi, secreted metabolites are one of the components that drive one-electron reductions of Fe(3+) and O2, generating Fenton chemistry reagents. Here we investigated whether such a mechanism is also implemented by P. involutus during organic matter decomposition. Activity-guided purification was performed to isolate the Fe(3+)-reducing principle secreted by P. involutus during growth on a maize compost extract. The Fe(3+)-reducing activity correlated with the presence of one compound. Mass spectrometry and nuclear magnetic resonance (NMR) identified this compound as the diarylcyclopentenone involutin. A major part of the involutin produced by P. involutus during organic matter decomposition was secreted into the medium, and the metabolite was not detected when the fungus was grown on a mineral nutrient medium. We also demonstrated that in the presence of H2O2, involutin has the capacity to drive an in vitro Fenton reaction via Fe(3+) reduction. Our results show that the mechanism for the reduction of Fe(3+) and the generation of hydroxyl radicals via Fenton chemistry by ectomycorrhizal fungi during organic matter decomposition is similar to that employed by the evolutionarily related brown rot saprotrophs during wood decay.

Correlation between pH dependence of O2 evolution and sensitivity of Mn cations in the oxygen-evolving complex to exogenous reductants.

Effects of pH, Ca(2+), and Cl(-) ions on the extraction of Mn cations from oxygen-evolving complex (OEC) in Ca-depleted photosystem II (PSII(-Ca)) by exogenous reductants hydroquinone (H2Q) and H2O2 were studied. Two of 4 Mn cations are released by H2Q and H2O2 at pHs 5.7, 6.5, and 7.5, and their extraction does not depend on the presence of Ca(2+) and Cl(-) ions. One of Mn cations ("resistant" Mn cation) cannot be extracted by H2Q and H2O2 at any pH. Extraction of 4th Mn ion ("flexible" Mn cation) is sensitive to pH, Ca(2+), and Cl(-). This Mn cation is released by reductants at pH 6.5 but not at pHs 5.7 and 7.5. A pH dependence curve of the oxygen-evolving activity in PSII(-Ca) membranes (in the presence of exogenous Ca(2+)) has a bell-shaped form with the maximum at pH 6.5. Thus, the increase in the resistance of flexible Mn cation in OEC to the action of reductants at acidic and alkaline pHs coincides with the decrease in oxygen evolution activity at these pHs. Exogenous Ca(2+) protects the extraction of flexible Mn cation at pH 6.5. High concentration of Cl(-) anions (100 mM) shifts the pH optimum of oxygen evolution to alkaline region (around pH 7.5), while the pH of flexible Mn extraction is also shifted to alkaline pH. This result suggests that flexible Mn cation plays a key role in the water-splitting reaction. The obtained results also demonstrate that only one Mn cation in Mn4 cluster is under strong control of calcium. The change in the flexible Mn cation resistance to exogenous reductants in the presence of Ca(2+) suggests that Ca(2+) can control the redox potential of this cation.

Tris(3-hydroxypropyl)phosphine is superior to dithiothreitol for in vitro assessment of vitamin K 2,3-epoxide reductase activity.

Use of the reductant dithiothreitol (DTT) as a substrate for measuring vitamin K 2,3-epoxide reductase (VKOR) activity in vitro has been reported to be problematic because it enables side reactions involving the vitamin K1 2,3-epoxide (K1>O) substrate. Here we characterize specific problems when using DTT and show that tris(3-hydroxypropyl)phosphine (THPP) is a reliable alternative to DTT for in vitro assessment of VKOR enzymatic activity. In addition, the pH buffering compound imidazole was found to be problematic in enhancing DTT-dependent non-enzymatic side reactions. Using THPP and phosphate-based pH buffering, we measured apparent Michaelis-Menten constants of 1.20 µM for K1>O and 260 µM for the active neutral form of THPP. The Km value for K1>O is in agreement with the value that we previously obtained using DTT (1.24 µM). Using THPP, we successfully eliminated non-enzymatic production of 3-hydroxyvitamin K1 and its previously reported base-catalyzed conversion to K1, both of which were shown to occur when DTT and imidazole are used as the reductant and pH buffer, respectively, in the in vitro VKOR assay. Accordingly, substitution of THPP for DTT in the in vitro VKOR assay will ensure more accurate enzymatic measurements and assessment of warfarin and other 4-hydroxycoumarin inhibition constants.

The autolysis of human HtrA1 is governed by the redox state of its N-terminal domain.

Human HtrA1 (high-temperature requirement protein A1) belongs to a conserved family of serine proteases involved in protein quality control and cell fate. The homotrimeric ubiquitously expressed protease has chymotrypsin-like specificity and primarily targets hydrophobic stretches in selected or misfolded substrate proteins. In addition, the enzyme is capable of exerting autolytic activity by removing the N-terminal insulin-like growth factor binding protein (IGFBP)/Kazal-like tandem motif without affecting the protease activity. In this study, we have addressed the mechanism governing the autolytic activity and find that it depends on the integrity of the disulfide bonds in the N-terminal IGFBP/Kazal-like domain. The specificity of the autolytic cleavage reveals a strong preference for cysteine in the P1 position of HtrA1, explaining the lack of autolysis prior to disulfide reduction. Significantly, the disulfides were reduced by thioredoxin, suggesting that autolysis of HtrA1 in vivo is linked to the endogenous redox balance and that the N-terminal domain acts as a redox-sensing switch.

A non-pediocin low molecular weight antimicrobial peptide produced by Pediococcus pentosaceus strain IE-3 shows increased activity under reducing environment.

BACKGROUND: Species of the genus Pediococcus are known to produce antimicrobial peptides such as pediocin-like bacteriocins that contain YGNGVXC as a conserved motif at their N-terminus. Until now, the molecular weight of various bacteriocins produced by different strains of the genus Pediococcus have been found to vary between 2.7 to 4.6 kD. In the present study, we characterized an antimicrobial peptide produced by P. pentosaceus strain IE-3. RESULTS: Antimicrobial peptide was isolated and purified from the supernatant of P. pentosaceus strain IE-3 grown for 48 h using cation exchange chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC) techniques. While MALDI-TOF MS experiments determined the precise molecular mass of the peptide to be 1701.00 Da, the de novo sequence (APVPFSCTRGCLTHLV) of the peptide revealed no similarity with reported pediocins and did not contain the YGNGVXC conserved motif. Unlike pediocin-like bacteriocins, the low molecular weight peptide (LMW) showed resistance to different proteases. Moreover, peptide treated with reducing agent like dithiothreitol (DTT) exhibited increased activity against both Gram-positive and Gram-negative test strains in comparison to native peptide. However, peptide treated with oxidizing agent such as hydrogen peroxide (H2O2) did not show any antimicrobial activity. CONCLUSION: To our knowledge this is the lowest molecular weight peptide produced by members of the genus Pediococcus. The low molecular weight peptide shared amino acid arrangement with N-terminal sequence of Class IIa, pediocin-like bacteriocins and showed increased activity under reducing conditions. Antimicrobial peptides active under reduced conditions are valuable for the preservation of processed foods like meat, dairy and canned foods where low redox potential prevails.

Assessment of the phytochemical constituents and antioxidant activity of a bloom forming microalgae Euglena tuba.

BACKGROUND: Unstable generation of free radicals in the body are responsible for many degenerative diseases. A bloom forming algae Euglena tuba growing abundantly in the aquatic habitats of Cachar district in the state of Assam in North-East India was analysed for its phytochemical contents, antioxidant activity as well as free radical scavenging potentials. RESULTS: Based on the ability of the extract in ABTS•+ radical cation inhibition and Fe3+ reducing power, the obtained results revealed the prominent antioxidant activity of the algae, with high correlation coefficient of its TEAC values to the respective phenolic and flavonoid contents. The extract had shown its scavenging activity for different free radicals and 41.89 ± 0.41 µg/ml, 5.83 ± 0.07 µg/ml, 278.46 ± 15.02 µg/ml and 223.25 ± 4.19 µg/ml were determined as the IC50 values for hydroxyl, superoxide, nitric oxide and hypochlorous acid respectively, which are lower than that of the corresponding reference standards. The phytochemical analysis also revealed that the phenolics, flavonoids, alkaloids, tannins and carbohydrates are present in adequate amount in the extract which was confirmed by HPLC analysis. CONCLUSIONS: The results showed that 70% methanol extract of the algae possesses excellent antioxidant and free radical scavenging properties.

Compositional studies and biological activities of some mash bean (Vigna mungo (L.) Hepper) cultivars commonly consumed in Pakistan.

BACKGROUND: In recent years, the desire to adopt a healthy diet has drawn attention to legume seeds and food products derived from them. Mash bean is an important legume crop used in Pakistan however a systematic mapping of the chemical composition of mash bean seeds is lacking. Therefore seeds of four mash bean (Vigna mungo (L.) Hepper, family Leguminoseae) cultivars (NARC-Mash-1, NARC-Mash-2, NARC-Mash-3, NARC-Mash-97) commonly consumed in Pakistan have been analyzed for their chemical composition, antioxidant potential and biological activities like inhibition of formation of advanced glycation end products (AGE) activity and tyrosinase inhibition activity. RESULTS: The investigated cultivars varied in terms of biochemical composition to various extents. Mineral composition indicated potassium and zinc in highest and lowest amounts respectively, in all cultivars. The amino acid profile in protein of these cultivars suggested cysteine is present in lowest quantity in all cultivars while fatty acid distribution pattern indicated unsaturated fatty acids as major fatty acids in all cultivars. All cultivars were found to be rich source of tocopherols and sterols. Fourier transform infrared spectroscopy (FTIR) fingerprints of seed flour and extracts indicated major functional groups such as polysaccharides, lipids, amides, amines and amino acids. Results indicated that all investigated cultivars possessed appreciable antioxidant potential. CONCLUSIONS: All cultivars are rich source of protein and possess sufficient content of dietary fiber, a balanced amino acid profile, low saturated fatty acids and antioxidant capacity that rationalizes many traditional uses of seeds of this crop besides its nutritional importance. The collected data will be useful for academic and corporate researchers, nutritionists and clinical dieticians as well as consumers. If proper attention is paid, it may become an important export commodity and may fetch considerable foreign exchange for Pakistan.