Role of quinone reductases in extracellular redox cycling in lichenized ascomycetes

Our previous work showed that many lichenized Ascomycetes can generate hydroxyl radicals using quinone-based extracellular redox cycling. During cycling, hydroquinones must be formed and subsequently regenerated from quinones using a quinone reductase (QR). However, we also showed that no simple correlation exists between QR activity and rates of hydroxyl radical formation. To further investigate the role of QR in hydroxyl radical formation, three model lichen species, Leptogium furfuraceum, Lasallia pustulata and Peltigera membranacea were selected for further investigation. All possessed QR activity and could metabolize quinones, and both Leptogium furfuraceum and Lasallia pustulata actively produced hydroxyl radicals. By contrast, P. membranacea produced almost no hydroxyl radicals, and although the lichen readily metabolized quinones, no hydroquinone production was detected. Peltigera had laccase (LAC) activity that was c. 50 times higher than in the other two species, suggesting that LAC rapidly oxidizes the hydroquinones, preventing radical formation deriving from auto-oxidation. It appears that in some lichens hydroxyl radical formation is blocked by the presence of high redox enzyme activity. QR from P. didactyla was studied further and found to display similar properties to the enzyme from free-living fungi, although it possessed an unusually high molecular mass (c. 62 kDa).     

Differential regulation of wheat quinone reductases in response to powdery mildew infection

At least two types of quinone reductases are present in plants: (1) the ζ-crystallin-like quinone reductases (QR1, EC 1.6.5.5) that catalyze the univalent reduction of quinones to semiquinone radicals, and (2) the DT-diaphorase-like quinone reductases (QR2, EC 1.6.99.2) that catalyze the divalent reduction of quinones to hydroquinones. QR2s protect cells from oxidative stress by making the quinones available for conjugation, thereby releasing them from the superoxide-generating one electron redox cycling, catalyzed by QR1s. Two genes, putatively encoding a QR1 and a QR2, respectively, were isolated from an expressed sequence tag collection derived from the epidermis of a diploid wheat Triticum monococcum L. 24 h after inoculation with the powdery mildew fungus Blumeria graminis (DC) EO Speer f. sp. tritici Em. Marchal. Northern analysis and tissue-specific RT-PCR showed that TmQR1 was repressed while TmQR2 was induced in the epidermis during powdery mildew infection. Heterologous expression of TmQR2 in Escherichia coli confirmed that the gene encoded a functional, dicumarol-inhibitable QR2 that could use either NADH or NADPH as an electron donor. The localization of dicumarol-inhibitable QR2 activity around powdery mildew infection sites was accomplished using a histochemical technique, based on tetrazolium dye reduction.     

Inhibition study of rabbit liver cytosolic reductases involved in daunorubicin toxication

Anthracycline cardiotoxicity represents the most unfavorable side effect of these highly efficient anticancer drugs. Several biotransformation enzymes have been described to contribute to their cardiotoxicity. Besides the activities of CYP450 isoforms which lead to the generation of reactive oxygen species (ROS), the cytosolic reductases have attracted attention nowadays. The reductases known to metabolize anthracyclines to C13-hydroxyanthracyclines are carbonyl reductase (CR, 1.1.1.184) and the aldo-keto reductases (AKR1C2, 1.3.1.20; AKR1A1, 1.1.1.2). Their participation in the formation of the toxic C13-hydroxymetabolite has been investigated in rabbit using diagnostic inhibitors of CR and AKR1C2. The kinetics and the type of reductase inhibition exerted by the two inhibitors have been described and it was found that CR was the main daunorubicin reductase at both optimal and physiological pH with the kinetic parameters for daunorubicin reduction of Km = 17.01 +/- 1.98 microM and V(max) = 139.60 +/- 5.64 pcat/mg. The IC50 values for quercitrin and flufenamic acid were 5.45 +/- 1.37 microM and 3.68 +/- 1.58 microM, respectively. The inhibition was uncompetitive for both inhibitors and irreversible in the case of flufenamic acid.     

Relationship of dithiothreitol-dependent microsomal vitamin K quinone and vitamin K epoxide reductases inhibition of epoxide reduction by vitamin K quinone

Vitamin K quinone was shown to be an effective inhibitor of vitamin K epoxide reduction by whole rat liver microsomes. Observation of inhibition was dependent upon the mode of addition of the substrate and inhibitor suggesting segregation of the compounds into different microsomal vesicles under certain conditions. The result is consistent with reduction of both vitamin K quinone and vitamin K epoxide by a single enzyme or a multisite enzyme complex.     

A novel cytosolic NADH:quinone oxidoreductase from Methanothermobacter marburgensis

Methanothermobacter marburgensis is a strictly anaerobic, thermophilic methanogenic archaeon that uses methanogenesis to convert H₂ and CO₂ to energy. M. marburgensis is one of the best-studied methanogens, and all genes required for methanogenic metabolism have been identified. Nonetheless, the present study describes a gene (Gene ID 9704440) coding for a putative NAD(P)H:quinone oxidoreductase that has not yet been identified as part of the metabolic machinery. The gene product, MmNQO, was successfully expressed, purified and characterized biochemically, as well as structurally. MmNQO was identified as a flavin-dependent NADH:quinone oxidoreductase with the capacity to oxidize NADH in the presence of a wide range of electron acceptors, whereas NADPH was oxidized with only three acceptors. The 1.50 Å crystal structure of MmNQO features a homodimeric enzyme where each monomer comprises 196 residues folding into flavodoxin-like α/β domains with non-covalently bound FMN (flavin mononucleotide). The closest structural homologue is the modulator of drug activity B from Streptococcus mutans with 1.6 Å root-mean-square deviation on 161 Cα atoms and 28% amino-acid sequence identity. The low similarity at sequence and structural level suggests that MmNQO is unique among NADH:quinone oxidoreductases characterized to date. Based on preliminary bioreactor experiments, MmNQO could provide a useful tool to prevent overflow metabolism in applications that require cells with high energy demand.     

Comparative analysis of cytosolic and solubilized mitochondrial menadione reductases from rat liver]

The kinetic properties of cytosolic and solubilized mitochondrial menadione reductases (EC 1.6.99.2) from rat liver were compared. The mechanism of the reaction of cytosolic and mitochondrial menadione reductases with NADH and 4-anilino-5-methoxy-1,2-benzoquinone (AMOBQ) as substrates obeys the "ping-pong" kinetics. AMOBQ is a competitive inhibitor of cytosolic menadione reductase (Ki = 219 microM). Both menadione reductases have similar or identical values of true and effective kinetic constants and similar electrophoretic mobilities.     

The known purified mammalian 2,4-dienoyl-CoA reductases are mitochondrial isoenzymes

The aim of this work was to determine the subcellular location of mammalian 2,4-dienoyl-CoA reductase, a key enzyme for degradation of polyunsaturated fatty acids by beta-oxidation. The enzyme was purified according to Kimura et al. (J Biochem 96:1463, 1984), and antibodies were raised in rabbits. Monospecific antibodies were obtained via purification on an affinity column. Immunoblotting of isolated rat liver mitochondria and peroxisomes with the monospecific reductase antibody showed that the antigen was located only in mitochondria. Immunocytochemical experiments with liver tissue, using the protein A-gold labeling technique, confirmed this result. The similarity of their characteristics suggests that the purified reductases described in the literature are the same isoenzyme. Consequently, since the rat enzyme was localized here to the mitochondria, purification and characterization of peroxisomal mammalian reductases remain to be achieved in the future. In addition, a significant induction also of mitochondrial reductase by clofibrate was observed in the immunoblotting experiments.     

Different catalytic mechanisms in mammalian selenocysteine- and cysteine-containing methionine-R-sulfoxide reductases

Selenocysteine (Sec) is found in active sites of several oxidoreductases in which this residue is essential for catalytic activity. However, many selenoproteins have fully functional orthologs, wherein cysteine (Cys) occupies the position of Sec. The reason why some enzymes evolve into selenoproteins if the Cys versions may be sufficient is not understood. Among three mammalian methionine-R-sulfoxide reductases (MsrBs), MsrB1 is a Sec-containing protein, whereas MsrB2 and MsrB3 contain Cys in the active site, making these enzymes an excellent system for addressing the question of why Sec is used in biological systems. In this study, we found that residues, which are uniquely conserved in Cys-containing MsrBs and which are critical for enzyme activity in MsrB2 and MsrB3, were not required for MsrB1, but increased the activity of its Cys mutant. Conversely, selenoprotein MsrB1 had a unique resolving Cys reversibly engaged in the selenenylsulfide bond. However, this Cys was not necessary for activities of either MsrB2, MsrB3, or the Cys mutant of MsrB1. We prepared Sec-containing forms of MsrB2 and MsrB3 and found that they were more than 100-fold more active than the natural Cys forms. However, these selenoproteins could not be reduced by the physiological electron donor, thioredoxin. Yet, insertion of the resolving Cys, which was conserved in MsrB1, into the selenoprotein form of MsrB3 restored the thioredoxin-dependent activity of this enzyme. These data revealed differences in catalytic mechanisms between selenoprotein MsrB1 and non-selenoproteins MsrB2 and MsrB3, and identified catalytic advantages and disadvantages of Sec- and Cys-containing proteins. The data also suggested that Sec- and Cys-containing oxidoreductases require distinct sets of active-site features that maximize their catalytic efficiencies and provide strategies for protein design with improved catalytic properties.     

Archaeal complex II: 'classical' and 'non-classical' succinate:quinone reductases with unusual features.

Reversible succinate dehydrogenase (SDH) activities have been ubiquitously detected in organisms from the three domains of life. They represent constituents either of respiratory complexes II in aerobes, or of fumarate dehydrogenase complexes in anaerobes. The present review gives a survey on archaeal succinate:quinone oxidoreductases (SQRs) analyzed so far. Though some of these could be studied in detail enzymologically and spectroscopically, the existence of others has been deduced only from published genome sequences. Interestingly, two groups of enzyme complexes can be distinguished in Archaea. One group resembles the properties of SDHs known from bacteria and mitochondria. The other represents a novel class with an unusual iron-sulfur cluster in subunit B and atypical sequence motifs in subunit C which may influence electron transport mechanisms and pathways. This novel class of SQRs is discussed in comparison to the so-called 'classical' complexes. A phylogenetic analysis is presented suggesting a co-evolution of the flavoprotein-binding subunit A and subunit B containing the three iron-sulfur clusters.     

Functional and structural aspects of poplar cytosolic and plastidial type a methionine sulfoxide reductases

The genome of Populus trichocarpa contains five methionine sulfoxide reductase A genes. Here, both cytosolic (cMsrA) and plastidial (pMsrA) poplar MsrAs were analyzed. The two recombinant enzymes are active in the reduction of methionine sulfoxide with either dithiothreitol or poplar thioredoxin as a reductant. In both enzymes, five cysteines, at positions 46, 81, 100, 196, and 202, are conserved. Biochemical and enzymatic analyses of the cysteine-mutated MsrAs support a catalytic mechanism involving three cysteines at positions 46, 196, and 202. Cys(46) is the catalytic cysteine, and the two C-terminal cysteines, Cys(196) and Cys(202), are implicated in the thioredoxin-dependent recycling mechanism. Inspection of the pMsrA x-ray three-dimensional structure, which has been determined in this study, strongly suggests that contrary to bacterial and Bos taurus MsrAs, which also contain three essential Cys, the last C-terminal Cys(202), but not Cys(196), is the first recycling cysteine that forms a disulfide bond with the catalytic Cys(46). Then Cys(202) forms a disulfide bond with the second recycling cysteine Cys(196) that is preferentially reduced by thioredoxin. In agreement with this assumption, Cys(202) is located closer to Cys(46) compared with Cys(196) and is included in a (202)CYG(204) signature specific for most plant MsrAs. The tyrosine residue corresponds to the one described to be involved in substrate binding in bacterial and B. taurus MsrAs. In these MsrAs, the tyrosine residue belongs to a similar signature as found in plant MsrAs but with the first C-terminal cysteine instead of the last C-terminal cysteine.     

Bacterial and mammalian HMG-CoA reductases: related enzymes with distinct architectures

The committed step in the biosynthesis of isoprenoids is catalyzed by HMG-CoA reductase. Recently determined structures of the distantly related bacterial and mammalian HMG-CoA reductases have revealed two distinct classes of the protein with differently formed active sites and oligomeric states. An appreciation of the remarkable differences in the active site architecture may ultimately lead to the development of novel antibacterial agents.     

Resolution and partial characterization of two aldehyde reductases of mammalian liver.

Investigation of NADP-dependent aldehyde reductase activity in mouse liver led to the finding that two distinct reductases are separable by DE52 ion exchange chromatography. Aldehyde reductase I (AR I) appears in the effluent, while aldehyde reductase II (AR II) is eluted with a salt gradient. By several procedures AR II was purified over 1100-fold from liver supernatant fraction, but AR I could be pruified only 107-fold because of its instability. The two enzymes are different in regard to pH optimum, substrate specificity, response to inhibitors, and reactivity with antibody to AR II. While both enzymes utilize aromatic aldehydes well, only AR II ACTS ON D-glucuronate, indicating that it is the aldyhyde reductase recently reported to be identical to NADP-L-gulonate dehydrogenase. The presence of two NADP-linked aldehyde reductases in liver has apparently not heretofore been reported.     

Specificity of hydrogen transfer of mammalian and avian carbonyl and aldehyde reductases

Aldehyde reductases from several mammalian and avian tissues transferred the pro-4R hydrogen of NADPH to the substrate, whereas the stereospecificity of carbonyl reductases was not uniform being correlated with the ability to catalyze the oxidoreduction of hydroxysteroids.     

Redox regulation of cell signaling by selenocysteine in mammalian thioredoxin reductases.

The intracellular generation of reactive oxygen species, together with the thioredoxin and glutathione systems, is thought to participate in redox signaling in mammalian cells. The activity of thioredoxin is dependent on the redox status of thioredoxin reductase (TR), the activity of which in turn is dependent on a selenocysteine residue. Two mammalian TR isozymes (TR2 and TR3), in addition to that previously characterized (TR1), have now been identified in humans and mice. All three TR isozymes contain a selenocysteine residue that is located in the penultimate position at the carboxyl terminus and which is encoded by a UGA codon. The generation of reactive oxygen species in a human carcinoma cell line was shown to result in both the oxidation of the selenocysteine in TR1 and a subsequent increase in the expression of this enzyme. These observations identify the carboxyl-terminal selenocysteine of TR1 as a cellular redox sensor and support an essential role for mammalian TR isozymes in redox-regulated cell signaling.     

Identification of two mammalian reductases involved in the two-carbon fatty acyl elongation cascade

The de novo synthesis of fatty acids occurs in two distinct cellular compartments. Palmitate (16:0) is synthesized from acetyl-CoA and malonyl-CoA in the cytoplasm by the enzymes acetyl-CoA carboxylase 1 and fatty acid synthase. The synthesis of fatty acids longer than 16 carbons takes place in microsomes and utilizes malonyl-CoA as the carbon source. Each two-carbon addition requires four sequential reactions: condensation, reduction, dehydration, and a final reduction to form the elongated fatty acyl-CoA. The initial condensation reaction is the regulated and rate-controlling step in microsomal fatty acyl elongation. We previously reported the cDNA cloning and characterization of a murine long chain fatty acyl elongase (LCE) . Overexpression of LCE in cells resulted in the enhanced addition of two-carbon units to C12-C16 fatty acids, and evidence was provided that LCE catalyzed the initial condensation reaction of long chain fatty acid elongation. The remaining three enzymes in the elongation reaction have not been identified in mammals. Here, we report the identification and characterization of two mammalian enzymes that catalyze the 3-ketoacyl-CoA and trans-2,3-enoyl-CoA reduction reactions in long and very long chain fatty acid elongation, respectively.