Rapid fluorescent visualization of multiple NAD(P)H oxidoreductases in homogenate,permeabilized cells,and tissue slices

Intracellular NAD(P)H oxidoreductases are a class of diverse enzymes that are the key players in a number of vital processes. The method we present and validate here is based on the ability of many NAD(P)H oxidoreductases to reduce the superoxide probe lucigenin, which is structurally similar to flavins, to its highly fluorescent water-insoluble derivative dimethylbiacridene. Two modifications of the method are proposed: (i) an express method for tissue homogenate and permeabilized cells in suspensions and (ii) a standard procedure for cells in culture and acute thin tissue slices. The method allows one to assess, visualize, and localize, using fluorescent markers of cellular compartments, multiple NADH and NADPH oxidoreductase activities. The application of selective inhibitors (e.g., VAS2870, a NOX2 inhibitor; plumbagin, a NOX4 inhibitor) allows one to distinguish and compare specific NAD(P)H oxidoreductase activities in cells and tissues and to attribute them to known enzymes. The method is simple, rapid, and flexible. It can be easily adapted to a variety of tasks. It will be useful for investigations of the role of various NAD(P)H oxidoreductases in a number of physiological and pathophysiological processes.     

New insights into type II NAD(P)H:quinone oxidoreductases.

Type II NAD(P)H:quinone oxidoreductases (NDH-2) catalyze the two-electron transfer from NAD(P)H to quinones, without any energy-transducing site. NDH-2 accomplish the turnover of NAD(P)H, regenerating the NAD(P)(+) pool, and may contribute to the generation of a membrane potential through complexes III and IV. These enzymes are usually constituted by a nontransmembrane polypeptide chain of approximately 50 kDa, containing a flavin moiety. There are a few compounds that can prevent their activity, but so far no general specific inhibitor has been assigned to these enzymes. However, they have the common feature of being resistant to the complex I classical inhibitors rotenone, capsaicin, and piericidin A. NDH-2 have particular relevance in yeasts like Saccharomyces cerevisiae and in several prokaryotes, whose respiratory chains are devoid of complex I, in which NDH-2 keep the balance and are the main entry point of electrons into the respiratory chains. Our knowledge of these proteins has expanded in the past decade, as a result of contributions at the biochemical level and the sequencing of the genomes from several organisms. The latter showed that most organisms contain genes that potentially encode NDH-2. An overview of this development is presented, with special emphasis on microbial enzymes and on the identification of three subfamilies of NDH-2.     

Rational proteomics I. Fingerprint identification and cofactor specificity in the short-chain oxidoreductase (SCOR) enzyme family

The short-chain oxidoreductase (SCOR) family of enzymes includes over 2000 members identified in sequenced genomes. Of these enzymes, approximately 200 have been characterized functionally, and the three-dimensional crystal structures of approximately 40 have been reported. Since some SCOR enzymes are involved in hypertension, diabetes, breast cancer, and polycystic kidney disease, it is important to characterize the other members of the family for which the biological functions are currently unknown. Although the SCOR family appears to have only a single fully conserved residue, it was possible, using bioinformatics methods, to determine characteristic fingerprints composed of 30-40 residues that are conserved at the 70% or greater level in SCOR subgroups. These fingerprints permit reliable prediction of several important structure-function features including NAD/NADP cofactor preference. For example, the correlation of aspartate or arginine residues with NAD or NADP binding, respectively, predicts the cofactor preference of more than 70% of the SCOR proteins with unknown function. The analysis of conserved residues surrounding the cofactor has revealed the presence of previously undetected CH em leader O hydrogen bonds in the majority of the SCOR crystal structures, predicts the presence of similar hydrogen bonds in 90% of the SCOR proteins of unknown function, and suggests that these hydrogen bonds may play a critical role in the catalytic functions of these enzymes.     

Nucleotide and deduced amino acid sequence of a human cDNA (NQO2) corresponding to a second member of the NAD(P)H:quinone oxidoreductase gene family. Extensive polymorphism at the NQO2 gene locus on chromosome 6.

NAD(P)H:quinone oxidoreductases (NQOs) are flavoproteins that catalyze the oxidation of NADH or NADPH by various quinones and oxidation-reduction dyes. We have previously described a complementary DNA that encodes a dioxin-inducible cytosolic form of human NAD(P)H:quinone oxidoreductase (NQO1). In the present report we describe the nucleotide sequence and deduced amino acid sequence for a cDNA clone that is likely to encode a second form of NAD(P)H:quinone oxidoreductase (NQO2) which was isolated by screening a human liver cDNA library by hybridization with a NQO1 cDNA probe. The NQO2 cDNA is 976 nucleotides long and encodes a protein of 231 amino acids (Mr = 25,956). The human NQO2 cDNA and protein are 54% and 49% similar to human liver cytosolic NQO1 cDNA and protein, respectively. COS1 cells transfected with NQO2 cDNA showed a 5-7-fold increase in NAD(P)H:quinone oxidoreductase activity as compared to nontransfected cells when either 2,6-dichlorophenolindophenol or menadione was used as substrate. Western blot analysis of the expressed NQO1 and NQO2 cDNA proteins showed cross-reactivity with rat NQO1 antiserum, indicating that NQO1 and NQO2 proteins are immunologically related. Northern blot analysis shows the presence of one NQO2 mRNA of 1.2 kb in control and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treated human hepatoblastoma Hep-G2 cells and that TCDD treatment does not lead to enhanced levels of NQO2 mRNA as it does for NQO1 mRNA. Southern blot analysis of human genomic DNA suggests the presence of a single gene approximately 14-17 kb in length. The NQO2 gene locus is highly polymorphic as indicated by several restriction fragment length polymorphisms detected with five different restriction enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Occurrence of hydroxysteroid oxidoreductases in liver of turtles

1. Hydroxysteroid oxidoreductases have been partially purified from the cytosol fraction (105,000 g supernatant) of liver from a fresh-water turtle (Podocnemis expansa) and a sea-water turtle (Chelonia mydas mydas) by precipitation with ammonium sulphate (AS, 10-80% saturation). 2. The following enzymes were detected (substrates in brackets): 3 alpha-hydroxysteroid oxidoreductase (androsterone), 3 beta-hydroxysteroid oxidoreductase (DHEA) and 17 beta-hydroxysteroid oxidoreductase (testosterone, oestradiol-17 beta). NAD as well as NADP were effective as cofactors. 3. In fresh-water turtle, highest activities of the 3 alpha-enzyme were measured in the 20% AS fraction (cofactor NAD), of the 3 beta-enzyme in the 60% AS fraction (cofactor NAD) and of the 17 beta-enzyme in the 40% AS fraction (cofactor NADP). 4. In sea-water turtle, highest activities were observed for all three enzymes in the 60% AS fraction. 5. Generally, enzyme activities were higher in sea-water turtles than in fresh-water turtles. The most active enzyme in both turtles was found to be the 3 alpha-hydroxysteroid oxidoreductase, followed by the 17 beta- and the 3 beta-hydroxysteroid oxidoreductases.     

Bacterial DNA ligases

DNA ligases join breaks in the phosphodiester backbone of DNA molecules and are used in many essential reactions within the cell. All DNA ligases follow the same reaction mechanism, but they may use either ATP or NAD+ as a cofactor. All Bacteria (eubacteria) contain NAD+-dependent DNA ligases, and the uniqueness of these enzymes to Bacteria makes them an attractive target for novel antibiotics. In addition to their NAD+-dependent enzymes, some Bacteria contain genes for putative ATP-dependent DNA ligases. The requirement for these different isozymes in Bacteria is unknown, but may be related to their utilization in different aspects of DNA metabolism. The putative ATP-dependent DNA ligases found in Bacteria are most closely related to proteins from Archaea and viruses. Phylogenetic analysis suggests that all NAD+-dependent DNA ligases are closely related, but the ATP-dependent enzymes have been acquired by Bacterial genomes on a number of separate occasions.     

Expansion and evolution of insect GMC oxidoreductases

Background  

The GMC oxidoreductases comprise a large family of diverse FAD enzymes that share a homologous backbone. The relationship and origin of the GMC oxidoreductase genes, however, was unknown. Recent sequencing of entire genomes has allowed for the evolutionary analysis of the GMC oxidoreductase family.     

NAD(P)依赖型氧化还原酶分离纯化技术进展

NAD(P)依赖型的氧化还原酶是一类重要的生物催化剂,在生物合成中被广泛应用。以亲和技术为基础的分离纯化方法与其它分离制备方法相比具有高选择性、高活力回收等优点。本文着重讨论亲和色谱技术在NAD(P)依赖型的氧化还原酶的分离纯化及制备中的研究进展。     

Sodium ion pumps and hydrogen production in glutamate fermenting anaerobic bacteria

Anaerobic bacteria ferment glutamate via two different pathways to ammonia, carbon dioxide, acetate, butyrate and molecular hydrogen. The coenzyme B12-dependent pathway in Clostridium tetanomorphum via 3-methylaspartate involves pyruvate:ferredoxin oxidoreductase and a novel enzyme, a membrane-bound NADH:ferredoxin oxidoreductase. The flavin- and iron-sulfur-containing enzyme probably uses the energy difference between reduced ferredoxin and NADH to generate an electrochemical Na+ gradient, which drives transport processes. The other pathway via 2-hydroxyglutarate in Acidaminococcus fermentans and Fusobacterium nucleatum involves glutaconyl-CoA decarboxylase, which uses the free energy of decarboxylation to generate also an electrochemical Na+ gradient. In the latter two organisms, similar membrane-bound NADH:ferredoxin oxidoreductases have been characterized. We propose that in the hydroxyglutarate pathway these oxidoreductases work in the reverse direction, whereby the reduction of ferredoxin by NADH is driven by the Na+ gradient. The reduced ferredoxin is required for hydrogen production and the activation of radical enzymes. Further examples show that reduced ferredoxin is an agent, whose reducing energy is about 1 ATP 'richer' than that of NADH.     

Reaction mechanism of azoreductases suggests convergent evolution with quinone oxidoreductases

Azoreductases are involved in the bioremediation by bacteria of azo dyes found in waste water. In the gut flora, they activate azo pro-drugs, which are used for treatment of inflammatory bowel disease, releasing the active component 5-aminosalycilic acid. The bacterium P. aeruginosa has three azoreductase genes, paAzoR1, paAzoR2 and paAzoR3, which as recombinant enzymes have been shown to have different substrate specificities. The mechanism of azoreduction relies upon tautomerisation of the substrate to the hydrazone form. We report here the characterization of the P. aeruginosa azoreductase enzymes, including determining their thermostability, cofactor preference and kinetic constants against a range of their favoured substrates. The expression levels of these enzymes during growth of P. aeruginosa are altered by the presence of azo substrates. It is shown that enzymes that were originally described as azoreductases, are likely to act as NADH quinone oxidoreductases. The low sequence identities observed among NAD(P)H quinone oxidoreductase and azoreductase enzymes suggests convergent evolution.     

AhpF and other NADH:peroxiredoxin oxidoreductases, homologues of low Mr thioredoxin reductase.

A group of bacterial flavoproteins related to thioredoxin reductase contain an additional approximately 200-amino-acid domain including a redox-active disulfide center at their N-termini. These flavoproteins, designated NADH:peroxiredoxin oxidoreductases, catalyze the pyridine-nucleotide-dependent reduction of cysteine-based peroxidases (e.g. Salmonella typhimurium AhpC, a member of the peroxiredoxin family) which in turn reduce H2O2 or organic hydroperoxides. These enzymes catalyze rapid electron transfer (kcat > 165 s-1) through one tightly bound FAD and two redox-active disulfide centers, with the N-terminal-most disulfide center acting as a redox mediator between the thioredoxin-reductase-like part of these proteins and the peroxiredoxin substrates. A chimeric protein with the first 207 amino acids of S. typhimurium AhpF attached to the N-terminus of Escherichia coli thioredoxin reductase exhibits very high NADPH:peroxiredoxin oxidoreductase and thioredoxin reductase activities. Catalytic turnover by NADH:peroxiredoxin oxidoreductases may involve major domain rotations, analogous to those proposed for bacterial thioredoxin reductase, and cycling of these enzymes between two electron-reduced (EH2) and four electron-reduced (EH4) redox states.     

Functional divergence between evolutionary-related LuxG and Fre oxidoreductases of luminous bacteria

In luminous bacteria NAD(P)H:flavin-oxidoreductases LuxG and Fre, there are homologous enzymes that could provide a luciferase with reduced flavin. Although Fre functions as a housekeeping enzyme, LuxG appears to be a source of reduced flavin for bioluminescence as it is transcribed together with luciferase. This study is aimed at providing the basic conception of Fre and LuxG evolution and revealing the peculiarities of the active site structure resulted from a functional variation within the oxidoreductase family. A phylogenetic analysis has demonstrated that Fre and LuxG oxidoreductases have evolved separately after the gene duplication event, and consequently, they have acquired changes in the conservation of functionally related sites. Namely, different evolutionary rates have been observed at the site responsible for specificity to flavin substrate (Arg 46). Also, Tyr 72 forming a part of a mobile loop involved in FAD binding has been found to be conserved among Fre in contrast to LuxG oxidoreductases. The conservation of different amino acid types in NAD(P)H binding site has been defined for Fre (arginine) and LuxG (proline) oxidoreductases.     

In vitro reconstitution of an NADPH-dependent superoxide reduction pathway from Pyrococcus furiosus

A scheme for the detoxification of superoxide in Pyrococcus furiosus has been previously proposed in which superoxide reductase (SOR) reduces (rather than dismutates) superoxide to hydrogen peroxide by using electrons from reduced rubredoxin (Rd). Rd is reduced with electrons from NAD(P)H by the enzyme NAD(P)H:rubredoxin oxidoreductase (NROR). The goal of the present work was to reconstitute this pathway in vitro using recombinant enzymes. While recombinant forms of SOR and Rd are available, the gene encoding P. furiosus NROR (PF1197) was found to be exceedingly toxic to Escherichia coli, and an active recombinant form (rNROR) was obtained via a fusion protein expression system, which produced an inactive form of NROR until cleavage. This allowed the complete pathway from NAD(P)H to the reduction of SOR via NROR and Rd to be reconstituted in vitro using recombinant proteins. rNROR is a 39.9-kDa protein whose sequence contains both flavin adenine dinucleotide (FAD)- and NAD(P)H-binding motifs, and it shares significant similarity with known and putative Rd-dependent oxidoreductases from several anaerobic bacteria, both mesophilic and hyperthermophilic. FAD was shown to be essential for activity in reconstitution assays and could not be replaced by flavin mononucleotide (FMN). The bound FAD has a midpoint potential of -173 mV at 23 degrees C (-193 mV at 80 degrees C). Like native NROR, the recombinant enzyme catalyzed the NADPH-dependent reduction of rubredoxin both at high (80 degrees C) and low (23 degrees C) temperatures, consistent with its proposed role in the superoxide reduction pathway. This is the first demonstration of in vitro superoxide reduction to hydrogen peroxide using NAD(P)H as the electron donor in an SOR-mediated pathway.     

Ca2+-binding and Ca2+-independent respiratory NADH and NADPH dehydrogenases of Arabidopsis thaliana

Type II NAD(P)H:quinone oxidoreductases are single polypeptide proteins widespread in the living world. They bypass the first site of respiratory energy conservation, constituted by the type I NADH dehydrogenases. To investigate substrate specificities and Ca(2+) binding properties of seven predicted type II NAD(P)H dehydrogenases of Arabidopsis thaliana we have produced them as T7-tagged fusion proteins in Escherichia coli. The NDB1 and NDB2 enzymes were found to bind Ca(2+), and a single amino acid substitution in the EF hand motif of NDB1 abolished the Ca(2+) binding. NDB2 and NDB4 functionally complemented an E. coli mutant deficient in endogenous type I and type II NADH dehydrogenases. This demonstrates that these two plant enzymes can substitute for the NADH dehydrogenases in the bacterial respiratory chain. Three NDB-type enzymes displayed distinct catalytic profiles with substrate specificities and Ca(2+) stimulation being considerably affected by changes in pH and substrate concentrations. Under physiologically relevant conditions, the NDB1 fusion protein acted as a Ca(2+)-dependent NADPH dehydrogenase. NDB2 and NDB4 fusion proteins were NADH-specific, and NDB2 was stimulated by Ca(2+). The observed activity profiles of the NDB-type enzymes provide a fundament for understanding the mitochondrial system for direct oxidation of cytosolic NAD(P)H in plants. Our findings also suggest different modes of regulation and metabolic roles for the analyzed A. thaliana enzymes.     

A path from primary protein sequence to ligand recognition

A novel method to organize protein structural information based solely on sequence is presented. The method clusters proteins into families that correlate with the three-dimensional protein structure and the conformation of the bound ligands. This procedure was applied to nicotinamide adenine dinucleotide [NAD(P)]-utilizing enzymes to identify a total of 94 sequence families, 53 of which are structurally characterized. Each of the structurally characterized proteins within a sequence family correlates to a single protein fold and to a common bound conformation of NAD(P). A wide range of structural folds is identified that recognize NAD(P), including Rossmann folds and beta/alpha barrels. The defined sequence families can be used to identify the type and prevalence of NAD(P)-utilizing enzymes in the proteomes of sequenced organisms. The proteome of Mycobacterium tuberculosis was mined to generate a proteome-wide profile of NAD(P)-utilizing enzymes coded by this organism. This enzyme family comprises approximately 6% of the open reading frames, with the largest subgroup being the Rossmann fold, short-chain dehydrogenases. The preponderance of short-chain dehydrogenases correlates strongly with the phenotype of M. tuberculosis, which is characterized as having one of the most complex prokaryotic cell walls.     

Putidaredoxin reductase,a new function for an old protein

Properties of recombinant wild type (WT) and six-histidine tag-fused (His(6)) putidaredoxin reductase (Pdr), a FAD-containing component of the soluble cytochrome P450cam monooxygenase system from Pseudomonas putida, have been studied. Both WT and His(6) Pdr were found to undergo a monomer-dimer association-dissociation and were partially present as an NAD(+)-bound form. Although molecular, spectral, and electron transferring properties of recombinant His(6) Pdr to artificial and native electron acceptors were similar to those of the WT protein, the presence of eight additional C-terminal amino acid residues, Pro-Arg-His-His-His-His-His-His, had a crucial effect on the enzyme interaction with oxidized pyridine nucleotide. Under anaerobic conditions, NAD(+) induced in His(6) Pdr spectral changes indicative of flavin reduction and formation of the charge transfer complex between the reduced FAD and NAD(+). The reaction proceeded considerably faster in the presence of free histidine and thiol-reducing agents, such as dithiothreitol and reduced glutathione. In the presence of any of these three reagents, NAD(+) was capable of inducing reduction of the flavin in WT Pdr. Free thiol groups were identified as an internal source of electrons in the enzyme. The results showed that WT and His(6) Pdr were able to function as NAD(H)-dependent dithiol/disulfide oxidoreductases catalyzing both forward and reverse reactions, NAD(+)-dependent oxidation of thiols, and NADH-dependent reduction of disulfides. This function of the flavoprotein can be dissociated from electron transfer to putidaredoxin. Similarity of Pdr to the enzymes of the glutathione reductase family is discussed.     

Mitochondrial Metabolism,Sirtuins, and Aging

The sirtuins are a family of proteins that act predominantly as nicotinamide adenine dinucleotide (NAD)-dependent deacetylases. In mammals seven sirtuin family members exist, including three members, Sirt3, Sirt4, and Sirt5, that localize exclusively within the mitochondria. Although originally linked to life-span regulation in simple organisms, this family of proteins appears to have various and diverse functions in higher organisms. One particular property that is reviewed here is the regulation of mitochondrial number, turnover, and activity by various mitochondrial and nonmitochondrial sirtuins. An emerging consensus from these recent studies is that sirtuins may act as metabolic sensors, using intracellular metabolites such as NAD and short-chain carbon fragments such as acetyl coenzyme A to modulate mitochondrial function to match nutrient supply.     

Flavogenomics--a genomic and structural view of flavin-dependent proteins

Riboflavin (vitamin B(2)) serves as the precursor for FMN and FAD in almost all organisms that utilize the redox-active isoalloxazine ring system as a coenzyme in enzymatic reactions. The role of flavin, however, is not limited to redox processes, as ～ 10% of flavin-dependent enzymes catalyze nonredox reactions. Moreover, the flavin cofactor is also widely used as a signaling and sensing molecule in biological processes such as phototropism and nitrogen fixation. Here, we present a study of 374 flavin-dependent proteins analyzed with regard to their function, structure and distribution among 22 archaeal, eubacterial, protozoan and eukaryotic genomes. More than 90% of flavin-dependent enzymes are oxidoreductases, and the remaining enzymes are classified as transferases (4.3%), lyases (2.9%), isomerases (1.4%) and ligases (0.4%). The majority of enzymes utilize FAD (75%) rather than FMN (25%), and bind the cofactor noncovalently (90%). High-resolution structures are available for about half of the flavoproteins. FAD-containing proteins predominantly bind the cofactor in a Rossmann fold (～ 50%), whereas FMN-containing proteins preferably adopt a (βα)(8)-(TIM)-barrel-like or flavodoxin-like fold. The number of genes encoding flavin-dependent proteins varies greatly in the genomes analyzed, and covers a range from ～ 0.1% to 3.5% of the predicted genes. It appears that some species depend heavily on flavin-dependent oxidoreductases for degradation or biosynthesis, whereas others have minimized their flavoprotein arsenal. An understanding of 'flavin-intensive' lifestyles, such as in the human pathogen Mycobacterium tuberculosis, may result in valuable new intervention strategies that target either riboflavin biosynthesis or uptake.     

PQQ and quinoprotein enzymes in microbial oxidations

Abstract Pyrroloquinoline quinone (PQQ) is found in a wide range of microorganisms, and several bacteria even excrete this compound into their culture medium when grown on alcohols. The existence of different classes of quinoprotein (PQQ-containing) enzymes is now well established (alcohol dehydrogenases, aldose (glucose) dehydrogenases, amine dehydrogenases and amine oxidases) while several other enzymes are suspected to be quinoproteins. In addition, many bacteria produce a quinoprotein apoenzyme, e.g., Escherichia coli and Pseudomonas testosteroni , producing glucose and ethanol dehydrogenase apoenzyme, respectively. It is unclear why these bacteria do not produce the holoenzyme form, but the apoenzymes have the ability to become functional, as was shown when the organisms were provided with PQQ. With this approach it could be demonstrated that E. coli has a non-phosphorylative route of glucose dissimilation via gluconate. Also, results with mixed cultures indicate that PQQ is a growth factor for certain bacteria under certain conditions. Despite the relatively high redox potential of the PQQ/PQQH₂ couple, quinoproteins transfer electrons to a variety of natural electron acceptors. Depending on the type of quinoprotein enzyme, the following components of the respiratory chain appear to be active: cytochrome c (sometimes with a copper protein as an intermediate), cytochrome b , and NADH dehydrogenase. PQQ is not restricted to a particular group of organisms, and reactions catalysed by quinoproteins can also be performed by NAD(P)-dependent or flavoprotein enzymes. Thus, these observations do not provide arguments for the view that quinoproteins have a unique role in microbial oxidations. Further comparative studies on oxidoreductases are necessary to reveal the special features of this novel group of enzymes.