Specific quinone reductase 2 inhibitors reduce metabolic burden and reverse Alzheimer's disease phenotype in mice.

Biological aging can be described as accumulative, prolonged metabolic stress and is the major risk factor for cognitive decline and Alzheimer's disease (AD). Recently, we identified and described a quinone reductase 2 (QR2) pathway in the brain, in which QR2 acts as a removable memory constraint and metabolic buffer within neurons. QR2 becomes overexpressed with age, and it is possibly a novel contributing factor to age-related metabolic stress and cognitive deficit. We found that, in human cells, genetic removal of QR2 produced a shift in the proteome opposing that found in AD brains while simultaneously reducing oxidative stress. We therefore created highly specific QR2 inhibitors (QR2is) to enable evaluation of chronic QR2 inhibition as a means to reduce biological age-related metabolic stress and cognitive decline. QR2is replicated results obtained by genetic removal of QR2, while local QR2i microinjection improved hippocampal and cortical-dependent learning in rats and mice. Continuous consumption of QR2is in drinking water improved cognition and reduced pathology in the brains of AD-model mice (5xFAD), with a noticeable between-sex effect on treatment duration. These results demonstrate the importance of QR2 activity and pathway function in the healthy and neurodegenerative brain and what we believe to be the great therapeutic potential of QR2is as first-in-class drugs.

Quinone binding site in a type VI sulfide:quinone oxidoreductase.

Monotopic membrane-bound flavoproteins, sulfide:quinone oxidoreductases (SQRs), have a variety of physiological functions, including sulfide detoxification. SQR enzymes are classified into six groups. SQRs use the flavin adenine dinucleotide (FAD) cofactor to transfer electrons from sulfide to quinone. A type VI SQR of the photosynthetic purple sulfur bacterium, Thiocapsa roseopersicina (TrSqrF), has been previously characterized, and the mechanism of sulfide oxidation has been proposed. This paper reports the characterization of quinone binding site (QBS) of TrSqrF composed of conserved aromatic and apolar amino acids. Val331, Ile333, and Phe366 were identified near the benzoquinone ring of enzyme-bound decylubiquinone (dUQ) using the TrSqrF homology model. In silico analysis revealed that Val331 and Ile333 alternately connected with the quinone head group via hydrogen bonds, and Phe366 and Trp369 bound the quinones via hydrophobic interactions. TrSqrF variants containing alanine (V331A, I333A, F366A) and aromatic amino acid (V331F, I333F, F366Y), as well as a C-terminal α-helix deletion (CTD) mutant were generated. These amino acids are critical for quinone binding and, thus, catalysis. Spectroscopic analyses proved that all mutants contained FAD. I333F replacement resulted in the lack of the charge transfer complex. In summary, the interactions described above maintain the quinone molecule's head in an optimal position for direct electron transfer from FAD. Surprisingly, the CTD mutant retained a relatively high level of specific activity while remaining membrane-anchored. This is a unique study because it focuses on the QBS and the oxidative stage of a type VI sulfide-dependent quinone reduction. KEY POINTS: • V331, I333, F366, and W369 were shown to interact with decylubiquinone in T. roseopersicina SqrF • These amino acids are involved in proper positioning of quinones next to FAD • I333 is essential in formation of a charge transfer complex from FAD to quinone.

Cloning, Expression, Purification, Crystallization, and X-Ray Structural Determination of the Human NQO2 in Complex with Melatonin.

Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone which possesses a wide range of biological effects. The effects mediated by melatonin are in part attributed to the antioxidant properties of the molecule, which may act as scavenger of free radicals, and also to the binding of melatonin to its protein targets. For a long time, melatonin had been described as a ligand of a putative "receptor" present in the mammalian brain. Several studies were thus carried out with the goal of clarifying the nature of this melatonin "receptor," which led to the discovery of MT3 as the third melatonin binding site. This binding site was confirmed independently by several groups, and it was eventually demonstrated that MT3 was the enzyme quinone reductase 2 (NQO2). Among the different approaches used to validate that MT3 was indeed NQO2, the co-crystallization of NQO2 with melatonin was key in demonstrating the exact binding site and mode of melatonin to the enzyme and led to a clear understanding of the residues important for protein binding and inhibition. In this chapter, we described the details for the cloning, expression, and purification of the human enzyme NQO2. We also describe a detailed protocol for the crystallization of melatonin with this protein.

Rhodaneses minimize the accumulation of cellular sulfane sulfur to avoid disulfide stress during sulfide oxidation in bacteria.

Heterotrophic bacteria and human mitochondria often use sulfide: quinone oxidoreductase (SQR) and persulfide dioxygenase (PDO) to oxidize sulfide to sulfite and thiosulfate. Bioinformatic analysis showed that the genes encoding RHOD domains were widely presented in annotated sqr-pdo operons and grouped into three types: fused with an SQR domain, fused with a PDO domain, and dissociated proteins. Biochemical evidence suggests that RHODs facilitate the formation of thiosulfate and promote the reaction between inorganic polysulfide and glutathione to produce glutathione polysulfide. However, the physiological roles of RHODs during sulfide oxidation by SQR and PDO could only be tested in an RHOD-free host. To test this, 8 genes encoding RHOD domains in Escherichia coli MG1655 were deleted to produce E. coli RHOD-8K. The sqrCp and pdoCp genes from Cupriavidus pinatubonensis JMP134 were cloned into E. coli RHOD-8K. SQRCp contains a fused RHOD domain at the N-terminus. When the fused RHOD domain of SQRCp was inactivated, the cells oxidized sulfide into increased thiosulfate with the accumulation of cellular sulfane sulfur in comparison with cells containing the intact sqrCp and pdoCp. The complementation of dissociated DUF442 minimized the accumulation of cellular sulfane sulfur and reduced the production of thiosulfate. Further analysis showed that the fused DUF442 domain modulated the activity of SQRCp and prevented it from directly passing the produced sulfane sulfur to GSH. Whereas, the dissociated DUF442 enhanced the PDOCp activity by several folds. Both DUF442 forms minimized the accumulation of cellular sulfane sulfur, which spontaneously reacted with GSH to produce GSSG, causing disulfide stress during sulfide oxidation. Thus, RHODs may play multiple roles during sulfide oxidation.

A redox cycle with complex II prioritizes sulfide quinone oxidoreductase-dependent H2S oxidation.

The dual roles of H2S as an endogenously synthesized respiratory substrate and as a toxin raise questions as to how it is cleared when the electron transport chain is inhibited. Sulfide quinone oxidoreductase (SQOR) catalyzes the first step in the mitochondrial H2S oxidation pathway, using CoQ as an electron acceptor, and connects to the electron transport chain at the level of complex III. We have discovered that at high H2S concentrations, which are known to inhibit complex IV, a new redox cycle is established between SQOR and complex II, operating in reverse. Under these conditions, the purine nucleotide cycle and the malate aspartate shuttle furnish fumarate, which supports complex II reversal and leads to succinate accumulation. Complex II knockdown in colonocytes decreases the efficiency of H2S clearance while targeted knockout of complex II in intestinal epithelial cells significantly decreases the levels of thiosulfate, a biomarker of H2S oxidation, to approximately one-third of the values seen in serum and urine samples from control mice. These data establish the physiological relevance of this newly discovered redox circuitry between SQOR and complex II for prioritizing H2S oxidation and reveal the quantitatively significant contribution of intestinal epithelial cells to systemic H2S metabolism.

Catalytic Control of Spiroketal Formation in Rubromycin Polyketide Biosynthesis.

The medically important bacterial aromatic polyketide natural products typically feature a planar, polycyclic core structure. An exception is found for the rubromycins, whose backbones are disrupted by a bisbenzannulated [5,6]-spiroketal pharmacophore that was recently shown to be assembled by flavin-dependent enzymes. In particular, a flavoprotein monooxygenase proved critical for the drastic oxidative rearrangement of a pentangular precursor and the installment of an intermediate [6,6]-spiroketal moiety. Here we provide structural and mechanistic insights into the control of catalysis by this spiroketal synthase, which fulfills several important functions as reductase, monooxygenase, and presumably oxidase. The enzyme hereby tightly controls the redox state of the substrate to counteract shunt product formation, while also steering the cleavage of three carbon-carbon bonds. Our work illustrates an exceptional strategy for the biosynthesis of stable chroman spiroketals.

Sulfide catabolism ameliorates hypoxic brain injury.

The mammalian brain is highly vulnerable to oxygen deprivation, yet the mechanism underlying the brain's sensitivity to hypoxia is incompletely understood. Hypoxia induces accumulation of hydrogen sulfide, a gas that inhibits mitochondrial respiration. Here, we show that, in mice, rats, and naturally hypoxia-tolerant ground squirrels, the sensitivity of the brain to hypoxia is inversely related to the levels of sulfide:quinone oxidoreductase (SQOR) and the capacity to catabolize sulfide. Silencing SQOR increased the sensitivity of the brain to hypoxia, whereas neuron-specific SQOR expression prevented hypoxia-induced sulfide accumulation, bioenergetic failure, and ischemic brain injury. Excluding SQOR from mitochondria increased sensitivity to hypoxia not only in the brain but also in heart and liver. Pharmacological scavenging of sulfide maintained mitochondrial respiration in hypoxic neurons and made mice resistant to hypoxia. These results illuminate the critical role of sulfide catabolism in energy homeostasis during hypoxia and identify a therapeutic target for ischemic brain injury.

Alteration of cofactor specificity of the acrylyl-CoA reductase from Escherichia coli.

OBJECTIVES: Alteration of the cofactor specificity of acrylyl-CoA reductase (AcuI) catalyzing the NAD(P)H-dependent reduction of acrylyl-CoA to propionyl-CoA is often desirable for designing of artificial metabolic pathways of various appointments. RESULTS: Several variants of AcuIs from Escherichia coli K-12 with multiple amino acid substitutions to alter the cofactor preference were obtained by site directed mutagenesis and the modified enzymes as His6-tagged proteins were characterized. The simultaneous substitutions of arginine-180, arginine-198 and serine-178 residues by alanine in the enzyme pocket sequence as well as other amino acid changes decreased both NADPH- and NADH-dependent activities in comparison to the wild-type enzyme. The replacement of serine-156 by glutamic acid decreased NADPH-dependent activity at least 7000-fold but NADH-dependent activity only by threefold. The replacement of serine-156 by aspartic acid decreased NADPH-dependent activity 70-fold with fair preservation of activity and specificity to NADH. CONCLUSIONS: These results demonstrated a relevance of Asp156 in the interaction of AcuI from E. coli K-12 with NADH as a coenzyme. These findings may provide reference information for shifting coenzyme specificity of acrylyl-CoA reductases.

A novel transcription factor MRPS27 up-regulates the expression of sqr, a key gene of mitochondrial sulfide metabolism in echiuran worm Urechis unicinctus.

Hydrogen sulfide is a natural, widely distributed, poisonous substance and sulfide: quinone oxidoreductase (SQR) is responsible for oxidizing hydrogen sulfide to less toxic sulfur compounds. The increase of SQR mRNA level is an important mechanism for organisms to adapt to hydrogen sulfide-rich environments. However, its transcriptional regulation mechanism is not very clear. In this study, a mitochondrial 28S ribosomal protein S27 (MRPS27), which has never been reported as a transcription factor, was screened by yeast one-hybrid experiment from the echiuran worm Urechis unicinctus, a benthic organism living in marine sediments. Western blotting indicated that UuMRPS27 contents increased significantly in the nuclear extract of hindgut under exposed to 150 µM sulfide. ChIP and EMSA assays demonstrated that UuMRPS27 did bind to the sqr proximal promoter, the key binding sequence was CTAGAG (+12 to +17 of the promoter) detected by DNase I footprinting assay as well as transient transfection experiments. Furthermore, UuMRPS27, as a transcription activator, exhibited the highest transcription activity compared with other reported sqr transcription factors. Our data revealed for the first time the role of MRPS27 acting as a transcription factor which expanded the understanding of sqr transcriptional regulation in sulfide metabolism mechanism.

Structural basis for persulfide-sensing specificity in a transcriptional regulator.

Cysteine thiol-based transcriptional regulators orchestrate the coordinated regulation of redox homeostasis and other cellular processes by 'sensing' or detecting a specific redox-active molecule, which in turn activates the transcription of a specific detoxification pathway. The extent to which these sensors are truly specific in cells for a singular class of reactive small-molecule stressors, for example, reactive oxygen or sulfur species, is largely unknown. Here, we report structural and mechanistic insights into the thiol-based transcriptional repressor SqrR, which reacts exclusively with oxidized sulfur species such as persulfides, to yield a tetrasulfide bridge that inhibits DNA operator-promoter binding. Evaluation of crystallographic structures of SqrR in various derivatized states, coupled with the results of a mass spectrometry-based kinetic profiling strategy, suggest that persulfide selectivity is determined by structural frustration of the disulfide form. These findings led to the identification of an uncharacterized repressor from the bacterial pathogen Acinetobacter baumannii as a persulfide sensor.

Insights into the catalytic mechanism of type VI sulfide:quinone oxidoreductases.

Sulfide oxidation is catalyzed by ancient membrane-bound sulfide:quinone oxidoreductases (SQR) which are classified into six different types. For catalysis of sulfide oxidation, all SQRs require FAD cofactor and a redox-active centre in the active site, usually formed between conserved essential cysteines. SQRs of different types have variation in the number and position of cysteines, highlighting the potential for diverse catalytic mechanisms. The photosynthetic purple sulfur bacterium, Thiocapsa roseopersicina contains a type VI SQR enzyme (TrSqrF) having unusual catalytic parameters and four cysteines likely involved in the catalysis. Site-directed mutagenesis was applied to identify the role of cysteines in the catalytic process of TrSqrF. Based on biochemical and kinetic characterization of these TrSqrF variants, Cys121 is identified as crucial for enzyme activity. The cofactor is covalently bound via a heterodisulfide bridge between Cys121 and the C8M group of FAD. Mutation of another cysteine present in all SQRs (Cys332) causes remarkably decreased enzyme activity (14.6% of wild type enzyme) proving important, but non-essential role of this residue in enzyme catalysis. The sulfhydril-blocking agent, iodoacetamide can irreversibly inactivate TrSqrF but only if substrates are present and the enzyme is actively catalyzing its reaction. When the enzyme is inhibited by iodoacetamide, the FAD cofactor is released. The inhibition studies support a mechanism that entails opening and reforming of the heterodisulfide bridge during the catalytic cycle of TrSqrF. Our study thus reports the first detailed structure-function analysis of a type VI SQR enzyme which enables the proposal of a distinct mechanism of sulfide oxidation for this class.

Identification of a gene encoding a novel thiosulfate:quinone oxidoreductase in marine Acidithiobacillus sp. strain SH.

Sulfur-oxidizing bacteria that are halophilic and acidophilic have gained interest because of their potential use in bioleaching operations in salt-containing environments. Acidithiobacillus sp. strain SH, which was previously identified as Acidithiobacillus thiooxidans based on its 16S rRNA gene sequence, is a chemolithoautotrophic marine bacterium exhibiting sodium chloride-stimulated thiosulfate-oxidizing activities. A novel thiosulfate:quinone oxidoreductase from strain SH (SH-TQO) has been purified from its solubilized membrane fraction. The gene for SH-TQO was determined from the draft genome sequence of the strain SH. Amino acid sequences of peptides generated by the in-gel trypsin digestion of SH-TQO were found in a protein encoded by locus tag B1757_09800 of the genome of the strain SH. The gene encoded 444 amino acids with a signal peptide of 29 amino acids and was annotated to encode a porin. The gene was located in a unique genomic region, not found in A. thiooxidans strains, suggesting that the strain SH acquired this region through a horizontal gene transfer. A protein-protein basic local alignment search revealed that sulfur-oxidizing bacteria, such as Acidithiobacillus species have proteins homologous to SH-TQO, though the degree of homologies was relatively low. The protein, DoxXA, which is homologous to TQO from Acidianus amvibalens, was also found in the genomic region.

Genetic and Biochemical Analysis of Anaerobic Respiration in Bacteroides fragilis and Its Importance In Vivo.

In bacteria, the respiratory pathways that drive molecular transport and ATP synthesis include a variety of enzyme complexes that utilize different electron donors and acceptors. This property allows them to vary the efficiency of energy conservation and to generate different types of electrochemical gradients (H+ or Na+). We know little about the respiratory pathways in Bacteroides species, which are abundant in the human gut, and whether they have a simple or a branched pathway. Here, we combined genetics, enzyme activity measurements, and mammalian gut colonization assays to better understand the first committed step in respiration, the transfer of electrons from NADH to quinone. We found that a model gut Bacteroides species, Bacteroides fragilis, has all three types of putative NADH dehydrogenases that typically transfer electrons from the highly reducing molecule NADH to quinone. Analyses of NADH oxidation and quinone reduction in wild-type and deletion mutants showed that two of these enzymes, Na+-pumping NADH:quinone oxidoreductase (NQR) and NADH dehydrogenase II (NDH2), have NADH dehydrogenase activity, whereas H+-pumping NADH:ubiquinone oxidoreductase (NUO) does not. Under anaerobic conditions, NQR contributes more than 65% of the NADH:quinone oxidoreductase activity. When grown in rich medium, none of the single deletion mutants had a significant growth defect; however, the double Δnqr Δndh2 mutant, which lacked almost all NADH:quinone oxidoreductase activity, had a significantly increased doubling time. Despite unaltered in vitro growth, the single nqr deletion mutant was unable to competitively colonize the gnotobiotic mouse gut, confirming the importance of NQR to respiration in B. fragilis and the overall importance of respiration to this abundant gut symbiont.IMPORTANCEBacteroides species are abundant in the human intestine and provide numerous beneficial properties to their hosts. The ability of Bacteroides species to convert host and dietary glycans and polysaccharides to energy is paramount to their success in the human gut. We know a great deal about the molecules that these bacteria extract from the human gut but much less about how they convert those molecules into energy. Here, we show that B. fragilis has a complex respiratory pathway with two different enzymes that transfer electrons from NADH to quinone and a third enzyme complex that may use an electron donor other than NADH. Although fermentation has generally been believed to be the main mechanism of energy generation in Bacteroides, we found that a mutant lacking one of the NADH:quinone oxidoreductases was unable to compete with the wild type in the mammalian gut, revealing the importance of respiration to these abundant gut symbionts.

Synechococcus sp. Strain PCC7002 Uses Sulfide:Quinone Oxidoreductase To Detoxify Exogenous Sulfide and To Convert Endogenous Sulfide to Cellular Sulfane Sulfur.

Eutrophication and deoxygenation possibly occur in coastal waters due to excessive nutrients from agricultural and aquacultural activities, leading to sulfide accumulation. Cyanobacteria, as photosynthetic prokaryotes, play significant roles in carbon fixation in the ocean. Although some cyanobacteria can use sulfide as the electron donor for photosynthesis under anaerobic conditions, little is known on how they interact with sulfide under aerobic conditions. In this study, we report that Synechococcus sp. strain PCC7002 (PCC7002), harboring an sqr gene encoding sulfide:quinone oxidoreductase (SQR), oxidized self-produced sulfide to S0, present as persulfide and polysulfide in the cell. The Δsqr mutant contained less cellular S0 and had increased expression of key genes involved in photosynthesis, but it was less competitive than the wild type in cocultures. Further, PCC7002 with SQR and persulfide dioxygenase (PDO) oxidized exogenous sulfide to tolerate high sulfide levels. Thus, SQR offers some benefits to cyanobacteria even under aerobic conditions, explaining the common presence of SQR in cyanobacteria.IMPORTANCE Cyanobacteria are a major force for primary production via oxygenic photosynthesis in the ocean. A marine cyanobacterium, PCC7002, is actively involved in sulfide metabolism. It uses SQR to detoxify exogenous sulfide, enabling it to survive better than its Δsqr mutant in sulfide-rich environments. PCC7002 also uses SQR to oxidize endogenously generated sulfide to S0, which is required for the proper expression of key genes involved in photosynthesis. Thus, SQR has at least two physiological functions in PCC7002. The observation provides a new perspective for the interplays of C and S cycles.

Comparison of sulfide-oxidizing Sulfurimonas strains reveals a new mode of thiosulfate formation in subsurface environments.

Sulfur-oxidizing Sulfurimonas spp. are widespread in sediments, hydrothermal vent fields, aquifers and subsurface environments such as oil reservoirs where they play an important role in the sulfur cycle. We determined the genome sequence of the oil field isolate Sulfurimonas sp. strain CVO and compared its gene expression during nitrate-dependent sulfide oxidation to the coastal sediment isolate Sulfurimonas denitrificans. Formation of elemental sulfur (S0 ) and high expression of sulfide quinone oxidoreductase (SQR) genes indicates that sulfide oxidation in both strains is mediated by SQR. Subsequent oxidation of S0 was achieved by the sulfur oxidation enzyme complex (SOX). In the coastal S. denitrificans, the genes are arranged and expressed as two clusters: soxXY1 Z1 AB and soxCDY2 Z2 H, and sulfate was the sole metabolic end product. By contrast, the oil field strain CVO has only the soxCDY2 Z2 H cluster and not soxXY1 Z1 AB. Despite the absence of the soxXY1 Z1 AB cluster, strain CVO oxidized S0 to thiosulfate and sulfate, demonstrating that soxCDY2 Z2 H genes alone are sufficient for S0 oxidation in Sulfurimonas spp. and that thiosulfate is an additional metabolic end product. Screening of publicly available metagenomes revealed that Sulfurimonas spp. with only the soxCDY2 Z2 H cluster are widespread suggesting this mechanism of thiosulfate formation is environmentally significant.

Impact of Na+-Translocating NADH:Quinone Oxidoreductase on Iron Uptake and nqrM Expression in Vibrio cholerae.

The Na+ ion-translocating NADH:quinone oxidoreductase (NQR) from Vibrio cholerae is a membrane-bound respiratory enzyme which harbors flavins and Fe-S clusters as redox centers. The NQR is the main producer of the sodium motive force (SMF) and drives energy-dissipating processes such as flagellar rotation, substrate uptake, ATP synthesis, and cation-proton antiport. The NQR requires for its maturation, in addition to the six structural genes nqrABCDEF, a flavin attachment gene, apbE, and the nqrM gene, presumably encoding a Fe delivery protein. We here describe growth studies and quantitative real-time PCR for the V. cholerae O395N1 wild-type (wt) strain and its mutant Δnqr and ΔubiC strains, impaired in respiration. In a comparative proteome analysis, FeoB, the membrane subunit of the uptake system for Fe2+ (Feo), was increased in V. choleraeΔnqr In this study, the upregulation was confirmed on the mRNA level and resulted in improved growth rates of V. choleraeΔnqr with Fe2+ as an iron source. We studied the expression of feoB on other respiratory enzyme deletion mutants such as the ΔubiC mutant to determine whether iron transport is specific to the absence of NQR resulting from impaired respiration. We show that the nqr operon comprises, in addition to the structural nqrABCDEF genes, the downstream apbE and nqrM genes on the same operon and demonstrate induction of the nqr operon by iron in V. cholerae wt. In contrast, expression of the nqrM gene in V. choleraeΔnqr is repressed by iron. The lack of functional NQR has a strong impact on iron homeostasis in V. cholerae and demonstrates that central respiratory metabolism is interwoven with iron uptake and regulation.IMPORTANCE Investigating strategies of iron acquisition, storage, and delivery in Vibrio cholerae is a prerequisite to understand how this pathogen thrives in hostile, iron-limited environments such as the human host. In addition to highlighting the maturation of the respiratory complex NQR, this study points out the influence of NQR on iron metabolism, thereby making it a potential drug target for antibiotics.

Cancer-associated variants of human NQO1: impacts on inhibitor binding and cooperativity.

Human NAD(P)H quinone oxidoreductase (DT-diaphorase, NQO1) exhibits negative cooperativity towards its potent inhibitor, dicoumarol. Here, we addressed the hypothesis that the effects of the two cancer-associated polymorphisms (p.R139W and p.P187S) may be partly mediated by their effects on inhibitor binding and negative cooperativity. Dicoumarol stabilized both variants and bound with much higher affinity for p.R139W than p.P187S. Both variants exhibited negative cooperativity towards dicoumarol; in both cases, the Hill coefficient (h) was approximately 0.5 and similar to that observed with the wild-type protein. NQO1 was also inhibited by resveratrol and by nicotinamide. Inhibition of NQO1 by resveratrol was approximately 10,000-fold less strong than that observed with the structurally similar enzyme, NRH quinine oxidoreductase 2 (NQO2). The enzyme exhibited non-cooperative behaviour towards nicotinamide, whereas resveratrol induced modest negative cooperativity (h = 0.85). Nicotinamide stabilized wild-type NQO1 and p.R139W towards thermal denaturation but had no detectable effect on p.P187S. Resveratrol destabilized the wild-type enzyme and both cancer-associated variants. Our data suggest that neither polymorphism exerts its effect by changing the enzyme's ability to exhibit negative cooperativity towards inhibitors. However, it does demonstrate that resveratrol can inhibit NQO1 in addition to this compound's well-documented effects on NQO2. The implications of these findings for molecular pathology are discussed.

Improvement of inhibitor tolerance in Saccharomyces cerevisiae by overexpression of the quinone oxidoreductase family gene YCR102C.

Budding yeast Saccharomyces cerevisiae is widely used for lignocellulosic biorefinery. However, its fermentation efficiency is challenged by various inhibitors (e.g. weak acids, furfural) in the lignocellulosic hydrolysate, and acetic acid is commonly present as a major inhibitor. The effects of oxidoreductases on the inhibitor tolerance of S. cerevisiae have mainly focused on furfural and vanillin, whereas the influence of quinone oxidoreductase on acetic acid tolerance is still unknown. In this study, we show that overexpression of a quinone oxidoreductase-encoding gene, YCR102C, in S. cerevisiae, significantly enhanced ethanol production under acetic acid stress as well as in the inhibitor mixture, and also improved resistance to simultaneous stress of 40°C and 3.6 g/L acetic acid. Increased catalase activities, NADH/NAD+ ratio and contents of several metals, especially potassium, were observed by YCR102C overexpression under acetic acid stress. To our knowledge, this is the first report that the quinone oxidoreductase family protein is related to acid stress tolerance. Our study provides a novel strategy to increase lignocellulosic biorefinery efficiency using yeast cell factory.

Hydrogen sulfide perturbs mitochondrial bioenergetics and triggers metabolic reprogramming in colon cells.

Unlike most other tissues, the colon epithelium is exposed to high levels of H2S derived from gut microbial metabolism. H2S is a signaling molecule that modulates various physiological effects. It is also a respiratory toxin that inhibits complex IV in the electron transfer chain (ETC). Colon epithelial cells are adapted to high environmental H2S exposure as they harbor an efficient mitochondrial H2S oxidation pathway, which is dedicated to its disposal. Herein, we report that the sulfide oxidation pathway enzymes are apically localized in human colonic crypts at the host-microbiome interface, but that the normal apical-to-crypt gradient is lost in colorectal cancer epithelium. We found that sulfide quinone oxidoreductase (SQR), which catalyzes the committing step in the mitochondrial sulfide oxidation pathway and couples to complex III, is a critical respiratory shield against H2S poisoning. H2S at concentrations ≤20 µm stimulated the oxygen consumption rate in colon epithelial cells, but, when SQR expression was ablated, H2S concentrations as low as 5 µm poisoned cells. Mitochondrial H2S oxidation altered cellular bioenergetics, inducing a reductive shift in the NAD+/NADH redox couple. The consequent electron acceptor insufficiency caused uridine and aspartate deficiency and enhanced glutamine-dependent reductive carboxylation. The metabolomic signature of this H2S-induced stress response mapped, in part, to redox-sensitive nodes in central carbon metabolism. Colorectal cancer tissues and cell lines appeared to counter the growth-restricting effects of H2S by overexpressing sulfide oxidation pathway enzymes. Our findings reveal an alternative mechanism for H2S signaling, arising from alterations in mitochondrial bioenergetics that drive metabolic reprogramming.

Kinetic Investigation of a Presumed Nitronate Monooxygenase from Pseudomonas aeruginosa PAO1 Establishes a New Class of NAD(P)H:Quinone Reductases.

PA0660 from Pseudomonas aeruginosa PAO1 is currently classified as a hypothetical nitronate monooxygenase (NMO), but no evidence at the transcript or protein level has been presented. In this study, PA0660 was purified and its biochemical and kinetic properties were characterized. Absorption spectroscopy and mass spectrometry demonstrated a tightly, noncovalently bound FMN in the active site of the enzyme. Analytical ultracentrifugation showed that the enzyme exists as a dimer in solution. Despite its annotation, PA0660 did not exhibit nitronate monooxygenase activity. The enzyme could be reduced with NADPH or NADH with a marked preference for NADPH, as indicated by ∼30-fold larger kcat/ Km and kred/ Kd values. Turnover could be sustained with NAD(P)H and quinones, DCPIP, and to a lesser extent molecular oxygen. However, PA0660 did not turn over with methyl red, consistent with a lack of azoreductase activity. The enzyme turned over through a ping-pong bi-bi steady-state kinetic mechanism with NADPH and 1,4-benzoquinone showing a kcat value of 90 s-1. The rate constant for flavin reduction with saturating NADPH was 360 s-1, whereas that for flavin oxidation with 1,4-benzoquinone was 270 s-1, consistent with both hydride transfers from the pyridine nucleotide to the flavin and from the flavin to 1,4-benzoquinone being partially rate-limiting for enzyme turnover. A BlastP search and a multiple-sequence alignment analysis of PA0660 highlighted the presence of six conserved motifs in >1000 open reading frames currently annotated as hypothetical NMOs. Our results suggest that PA0660 should be classified as an NAD(P)H:quinone reductase and serve as a paradigm enzyme for a new class of enzymes.