Mechanism of methylmercury release from bound type in bloodstream or tissues.

Reductants involving selenite freed methylmercury from bound protein without breaking the mercury-carbon bond in conjunction with sulfhydryls such as cysteine or homocysteine. The free methylmercury was transfered through an organic layer from the original -SH radicals to the other -SH radicals. The same effect occurred in normal physical reactions resulting from oxidoreductases, alcohol or lactic dehydrogenase. The oxidation-reduction system may play an important role in transfer of methylmercury from blood to tissues, or vice versa.     

The oxidation-reduction kinetics of cytochromes b, c1 and c in initially fully reduced mitochondrial membranes are in agreement with the Q-cycle hypothesis

Stopped-flow experiments were performed to distinguish between two hypotheses, the Q-cycle and the SQ-cycle, each describing the pathway of electron transfer in the QH2:cytochrome c oxidoreductases. It was observed that, when mitochondrial membranes from the yeast Saccharomyces cerevisiae were poised at a low redox potential with appropriate amounts of sodium dithionite to completely reduce cytochrome b, the kinetics of oxidation of cytochrome b showed a lag period of maximally 100 ms. Under the same experimental conditions, the oxidation-reduction kinetics of cytochromes c + c1 showed transient behaviour. These results do not support the presence of a mobile species of semiquinone in the QH2:cytochrome c oxidoreductases, as envisaged in the SQ-cycle, but are consistent with a Q-cycle mechanism in which the two quinone-binding domains do not exchange electrons directly on the timescale of turnover of the enzyme.     

Review of NAD(P)H-dependent oxidoreductases: Properties,engineering and application

NAD(P)H-dependent oxidoreductases catalyze the reduction or oxidation of a substrate coupled to the oxidation or reduction, respectively, of a nicotinamide adenine dinucleotide cofactor NAD(P)H or NAD(P)⁺. NAD(P)H-dependent oxidoreductases catalyze a large variety of reactions and play a pivotal role in many central metabolic pathways. Due to the high activity, regiospecificity and stereospecificity with which they catalyze redox reactions, they have been used as key components in a wide range of applications, including substrate utilization, the synthesis of chemicals, biodegradation and detoxification. There is great interest in tailoring NAD(P)H-dependent oxidoreductases to make them more suitable for particular applications. Here, we review the main properties and classes of NAD(P)H-dependent oxidoreductases, the types of reactions they catalyze, some of the main protein engineering techniques used to modify their properties and some interesting examples of their modification and application.     

NAD(P)H依赖型氧化还原酶不对称还原胺化制备手性胺的研究进展

不对称还原胺化反应是制备医药中间体手性胺结构单元的重要反应。目前已有许多不同种类的酶被应用于合成手性胺,其中NAD(P)H依赖型氧化还原酶催化的还原胺化反应最为引人注目,因为其能够一步将潜手性酮化合物完全转化为光学纯的手性胺化合物。文中以亚胺还原酶、氨基酸脱氢酶、冠瘿碱脱氢酶和还原性酮胺化酶为例,从NAD(P)H依赖型氧化还原酶的结构特征、作用机理、分子改造及催化应用等方面,综述了其在不对称还原胺化合成手性胺领域的研究进展。     

Alteration of enzyme specificity and catalysis.

In the past year, site-directed mutagenesis and other forms of protein engineering have been used to reverse the substrate specificity of several pairs of enzymes, including disulphide oxidoreductases, proteases, sugar-processing enzymes, and nucleases, as well as the specificity of hormones and their receptors. Mutations have been found that affect rate-determining steps, allowing normally transient intermediates to accumulate. Other mutations endow enzymes with totally new chemical reactions, and even novel biological functions. A combination of molecular genetics and chemical modification has been used for protein engineering.     

High-fat diet causes iron deficiency via hepcidin-independent reduction of duodenal iron absorption

Obesity is often associated with disorders of iron homeostasis; however, the underlying mechanisms are not fully understood. Hepcidin is a key regulator of iron metabolism and may be responsible for obesity-driven iron deficiency. Herein, we used an animal model of diet-induced obesity to study high-fat-diet-induced changes in iron homeostasis. C57BL/6 mice were fed a standard (SD) or high-fat diet (HFD) for 8 weeks, and in addition, half of the mice received high dietary iron (Fe+) for the last 2 weeks. Surprisingly, HFD led to systemic iron deficiency which was traced back to reduced duodenal iron absorption. The mRNA and protein expressions of the duodenal iron transporters Dmt1 and Tfr1 were significantly higher in HFD- than in SD-fed mice, indicating enterocyte iron deficiency, whereas the mRNA levels of the duodenal iron oxidoreductases Dcytb and hephaestin were lower in HFD-fed mice. Neither hepatic and adipose tissue nor serum hepcidin concentrations differed significantly between SD- and HFD-fed mice, whereas dietary iron supplementation resulted in increased hepatic hepcidin mRNA expression and serum hepcidin levels in SD as compared to HFD mice. Our study suggests that HFD results in iron deficiency which is neither due to intake of energy-dense nutrient poor food nor due to increased sequestration in the reticulo-endothelial system but is the consequence of diminished intestinal iron uptake. We found that impaired iron absorption is independent of hepcidin but rather results from reduced metal uptake into the mucosa and discordant oxidoreductases expressions despite enterocyte iron deficiency.     

Dramatic down-regulation of oxidoreductases in human hepatocellular carcinoma hepG2 cells: proteomics and gene ontology unveiling new frontiers in cancer enzymology

Background

Oxidoreductases are enzymes that catalyze many redox reactions in normal and neoplastic cells. Their actions include catalysis of the transformation of free, neutral oxygen gas into oxygen free radicals, superoxide, hydroperoxide, singlet oxygen and hydrogen peroxide. These activated forms of oxygen contribute to oxidative stress that modifies lipids, proteins, DNA and carbohydrates. On the other hand, oxidoreductases constitute one of the most important free radical scavenger systems typified by catalase, superoxide dismutase and glutathione peroxidase. In this work, proteomics, Gene Ontology mapping and Directed Acyclic Graphs (DAG) are employed to detect and quantify differential oxidoreductase enzyme expressions between HepG2 cells and normal human liver tissues.

Results

For the set of bioinformatics calculations whose BLAST searches are performed using the BLAST program BLASTP 2.2.13 [Nov-27-2005], DAG of the Gene Ontology's Molecular Function annotations show that oxidoreductase activity parent node of the liver proteome contains 331 annotated protein sequences, 7 child nodes and an annotation score of 188.9, whereas that of HepG2 cells has 188 annotated protein sequences, 3 child nodes and an annotation score of only 91.9. Overwhelming preponderance of oxidoreductases in the liver is additionally supported by the isomerase DAGs: nearly all the reactions described in the normal liver isomerase DAG are oxidoreductase isomerization reactions, whereas only one of the three child nodes in the HepG2 isomerase DAG is oxidoreductase. Upon normalization of the annotation scores to the parent Molecular Function nodes, oxidoreductases are down-regulated in HepG2 cells by 58%. Similarly, for the set of bioinformatics calculations whose BLAST searches are carried out using BLASTP 2.2.15 [Oct-15-2006], oxidoreductases are down-regulated in HepG2 cells by 56%.

Conclusion

Proteomics and Gene Ontology reveal, for the first time, differential enzyme activities between HepG2 cells and normal human liver tissues, which may be a promising new prognostic marker of Hepatocellular carcinoma. Two independent sets of bioinformatics calculations that employ two BLAST program versions, and searched different databases, arrived at essentially the same conclusion: oxidoreductases are down-regulated in HepG2 cells by approximately 57%, when compared to normal human liver tissues. Down-regulation of oxidoreductases in hepatoma is additionally supported by Gene Ontology analysis of isomerises.     

Oxidoreductases on their way to industrial biotransformations

Fungi produce heme-containing peroxidases and peroxygenases, flavin-containing oxidases and dehydrogenases, and different copper-containing oxidoreductases involved in the biodegradation of lignin and other recalcitrant compounds. Heme peroxidases comprise the classical ligninolytic peroxidases and the new dye-decolorizing peroxidases, while heme peroxygenases belong to a still largely unexplored superfamily of heme-thiolate proteins. Nevertheless, basidiomycete unspecific peroxygenases have the highest biotechnological interest due to their ability to catalyze a variety of regio- and stereo-selective monooxygenation reactions with H₂O₂ as the source of oxygen and final electron acceptor. Flavo-oxidases are involved in both lignin and cellulose decay generating H₂O₂ that activates peroxidases and generates hydroxyl radical. The group of copper oxidoreductases also includes other H₂O₂ generating enzymes - copper-radical oxidases - together with classical laccases that are the oxidoreductases with the largest number of reported applications to date. However, the recently described lytic polysaccharide monooxygenases have attracted the highest attention among copper oxidoreductases, since they are capable of oxidatively breaking down crystalline cellulose, the disintegration of which is still a major bottleneck in lignocellulose biorefineries, along with lignin degradation. Interestingly, some flavin-containing dehydrogenases also play a key role in cellulose breakdown by directly/indirectly “fueling” electrons for polysaccharide monooxygenase activation. Many of the above oxidoreductases have been engineered, combining rational and computational design with directed evolution, to attain the selectivity, catalytic efficiency and stability properties required for their industrial utilization. Indeed, using ad hoc software and current computational capabilities, it is now possible to predict substrate access to the active site in biophysical simulations, and electron transfer efficiency in biochemical simulations, reducing in orders of magnitude the time of experimental work in oxidoreductase screening and engineering. What has been set out above is illustrated by a series of remarkable oxyfunctionalization and oxidation reactions developed in the frame of an intersectorial and multidisciplinary European RTD project. The optimized reactions include enzymatic synthesis of 1-naphthol, 25-hydroxyvitamin D₃, drug metabolites, furandicarboxylic acid, indigo and other dyes, and conductive polyaniline, terminal oxygenation of alkanes, biomass delignification and lignin oxidation, among others. These successful case stories demonstrate the unexploited potential of oxidoreductases in medium and large-scale biotransformations.     

Evolving thermostability in mutant libraries of ligninolytic oxidoreductases expressed in yeast

Background  

In the picture of a laboratory evolution experiment, to improve the thermostability whilst maintaining the activity requires of suitable procedures to generate diversity in combination with robust high-throughput protocols. The current work describes how to achieve this goal by engineering ligninolytic oxidoreductases (a high-redox potential laccase -HRPL- and a versatile peroxidase, -VP-) functionally expressed in Saccharomyces cerevisiae.     

Recent biotechnological developments in the use of peroxidases

Peroxidases are ubiquitous oxidoreductases that use hydrogen peroxide or alkyl peroxides as oxidants. Advances have recently been made in using them to prepare, under mild and controlled conditions, chiral organic molecules that are valuable for the chemoenzymatic synthesis of a wide range of useful compounds. Horseradish peroxidase can be converted into a peroxygenative enzyme by molecular engineering. Chloroperoxidase, the most versatile peroxidase, behaves like a 'true' monooxygenase in sulfoxidations with molecular oxygen and an external reductant, with substantial increases in enantioselectivity and enzyme stability.     

Dioxygenase- and monooxygenase-catalysed synthesis of <Emphasis Type="Italic">cis</Emphasis>-dihydrodiols,catechols, epoxides and other oxygenated products

Oxidoreductases are an emerging class of biotechnologically relevant enzymes due to their regio- and stereo-specificity. The selective oxygenation of aromatic compounds by oxidoreductases has received much attention and a wide range of reactions have been documented using these enzymes from various microbial sources. This review gives an overview of various dioxygenase, monooxygenase and oxidase enzymes that have been manipulated for the synthesis of products such as cis-dihydrodiols, catechols, epoxides and other oxygenated products. The use of protein engineering and its advancement in the synthesis of recombinant enzymes is also discussed.     

Ero1-α and PDIs constitute a hierarchical electron transfer network of endoplasmic reticulum oxidoreductases

Ero1-α and endoplasmic reticulum (ER) oxidoreductases of the protein disulfide isomerase (PDI) family promote the efficient introduction of disulfide bonds into nascent polypeptides in the ER. However, the hierarchy of electron transfer among these oxidoreductases is poorly understood. In this paper, Ero1-α–associated oxidoreductases were identified by proteomic analysis and further confirmed by surface plasmon resonance. Ero1-α and PDI were found to constitute a regulatory hub, whereby PDI induced conformational flexibility in an Ero1-α shuttle cysteine (Cys99) facilitated intramolecular electron transfer to the active site. In isolation, Ero1-α also oxidized ERp46, ERp57, and P5; however, kinetic measurements and redox equilibrium analysis revealed that PDI preferentially oxidized other oxidoreductases. PDI accepted electrons from the other oxidoreductases via its a′ domain, bypassing the a domain, which serves as the electron acceptor from reduced glutathione. These observations provide an integrated picture of the hierarchy of cooperative redox interactions among ER oxidoreductases in mammalian cells.     

Diversity and origin of alternative NADH:ubiquinone oxidoreductases

Mitochondria from various organisms, especially plants, fungi and many bacteria contain so-called alternative NADH:ubiquinone oxidoreductases that catalyse the same redox reaction as respiratory chain complex I, but do not contribute to the generation of transmembrane proton gradients. In eucaryotes, these enzymes are associated with the mitochondrial inner membrane, with their NADH reaction site facing either the mitochondrial matrix (internal alternative NADH:ubiquinone oxidoreductases) or the cytoplasm (external alternative NADH:ubiquinone oxidoreductases). Some of these enzymes also accept NADPH as substrate, some require calcium for activity. In the past few years, the characterisation of several alternative NADH:ubiquinone oxidoreductases on the DNA and on the protein level, of substrate specificities, mitochondrial import and targeting to the mitochondrial inner membrane has greatly improved our understanding of these enzymes. The present review will, with an emphasis on yeast model systems, illuminate various aspects of the biochemistry of alternative NADH:ubiquinone oxidoreductases, address recent developments and discuss some of the questions still open in the field.     

Potential role of thiol:disulfide oxidoreductases in the pathogenesis of Helicobacter pylori

Helicobacter pylori infections are responsible for a sequence of molecular events which ultimately result in the development of gastric diseases. The pathogenesis of H. pylori has been studied extensively with strong focus on the identification of virulence factors. In contrast, the involvement of thiol:disulfide oxidoreductases in bacterial pathogenesis is less well understood. This paper provides a review of the current knowledge of H. pylori putative thiol:disulfide oxidoreductases, and their potential role in promoting virulence and colonization. Several bioinformatic analyses served to complete the information on these oxidoreductases of H. pylori.     

Glutathione directly reduces an oxidoreductase in the endoplasmic reticulum of mammalian cells

The formation of disulfide bonds is an essential step in the folding of many glycoproteins and secretory proteins. Non-native disulfide bonds are often formed between incorrect cysteine residues, and thus the cell has dedicated a family of oxidoreductases that are thought to isomerize non-native bonds. For an oxidoreductase to be capable of performing isomerization or reduction reactions, it must be maintained in a reduced state. Here we show that most of the oxidoreductases are predominantly reduced in vivo. Following oxidative stress the oxidoreductases are quickly reduced, demonstrating that a robust reductive pathway is in place in mammalian cells. Using ERp57 as a model we show that the reductive pathway is cytosol-dependent and that the component responsible for the reduction of the oxidoreductases is the low molecular mass thiol glutathione. In addition, ERp57 is not reduced following oxidative stress when inhibitors of glutathione synthesis or glutathione reduction are added to cells. Glutathione directly reduces ERp57 at physiological concentrations in vitro, and biotinylated glutathione forms a mixed disulfide with ERp57 in microsomes. Our results demonstrate that glutathione plays a direct role in the isomerization of disulfide bonds by maintaining the mammalian oxidoreductases in a reduced state.     

Flow cytometry-based assay for the activity of NAD(P)H oxidoreductases of the outer mitochondrial membrane

NAD(P)H oxidoreductases of the outer mitochondrial membrane (OMM) are able to activate various xenobiotics and stimulate the production of reactive oxygen species and the opening of the mitochondrial permeability transition pore. However, the role of these systems in the cell damage by xenobiotics and chemotherapeutic drugs is poorly understood because the methods for the selective assessment of their activity have not been elaborated and specific inhibitors are unknown. Here we propose a method for the semiquantitative assessment of the activity of NAD(P)H oxidoreductases of the OMM in intact and permeabilized cells that is based on the flow cytometry detection of dimethylbiacridene, a fluorescent product of two-electron reduction of lucigenin. The method uses the structural feature of mitochondrial organization: the proximity of the sites of one-electron reduction of lucigenin to cation radical (NAD(P)H oxidoreductases of the OMM) to the sites of its subsequent oxidation (cytochrome c oxidase). The inhibition of cytochrome c oxidase by cyanide selectively activates the dimethylbiacridene formation by oxidoreductases of the OMM but not by other cellular oxidoreductases. The proposed protocol allows one to assess the lucigenin reductase (two-electron) activity of NAD(P)H oxidoreductases of the OMM and to compare it with the activity of other cellular systems that can be used for the analysis of the role of these systems in the cell damage by xenobiotics and antitumor drugs.     

细菌中倍半萜生物合成的研究进展

萜类化合物是一大类小分子天然产物,在生物体内扮演重要的角色。植物和真菌中萜类化合物的生物合成已被广泛研究,但是在真核生物中克隆或改造萜类化合物生物合成途径还有较大难度。许多细菌同样可以产生萜类化合物。在过去十多年间细菌萜类合酶的研究进展为我们对萜类化合物生物合成的理解做出了显著的贡献。这里我们主要关注细菌中合成的倍半萜化合物,概述其化学结构、倍半萜合酶对法尼基焦磷酸环化的机制、后修饰酶特别是氧化还原酶所参与的后修饰、代谢调控以及合成途径中尚未解决的问题等。     

Studies of the control of luminescence in Beneckea harveyi: properties of the NADH and NADPH:FMN oxidoreductases.

Highly purified NADH and NADPH:FMN oxidoreductases from Beneckea harveyi have been characterized with regard to kinetic parameters, association with luciferase, activity with artificial electron acceptors, and the effects of inhibitors. The NADH:FMN oxidoreductase exhibits single displacement kinetics while the NADPH:FMN oxidoreductase exhibits double displacement or ping-pong kinetics. This is consistent with the formation of a reduced enzyme as an intermediate in the reaction of catalyzed by the NADPH:FMN oxidoreductase. Coupling of either of the oxidoreductases to the luciferase reaction decreases the apparent Kms for NADH, NADPH, and FMN, supporting the suggestion of a complex between the oxidoreductases and luciferase. The soluble oxidoreductases are more efficient in producing light with luciferase than is a NADH dehydrogenase preparation obtained from the membranes of these bacteria. The soluble enzymes use either FMN or FAD as substrates for the oxidation of reduced pyridine nucleotides while the membrane NADH dehydrogenase is much more active with artificial electron acceptors such as ferricyanide and methylene blue. FMN and FAD are very poor acceptors. The evidence indicates that neither of the soluble oxidoreductases is derived from the membranes. Both enzymes are constitutive and do not depend on the synthesis of luciferase.     

A Structure-Based Approach for Detection of Thiol Oxidoreductases and Their Catalytic Redox-Active Cysteine Residues

Cysteine (Cys) residues often play critical roles in proteins, for example, in the formation of structural disulfide bonds, metal binding, targeting proteins to the membranes, and various catalytic functions. However, the structural determinants for various Cys functions are not clear. Thiol oxidoreductases, which are enzymes containing catalytic redox-active Cys residues, have been extensively studied, but even for these proteins there is little understanding of what distinguishes their catalytic redox Cys from other Cys functions. Herein, we characterized thiol oxidoreductases at a structural level and developed an algorithm that can recognize these enzymes by (i) analyzing amino acid and secondary structure composition of the active site and its similarity to known active sites containing redox Cys and (ii) calculating accessibility, active site location, and reactivity of Cys. For proteins with known or modeled structures, this method can identify proteins with catalytic Cys residues and distinguish thiol oxidoreductases from the enzymes containing other catalytic Cys types. Furthermore, by applying this procedure to Saccharomyces cerevisiae proteins containing conserved Cys, we could identify the majority of known yeast thiol oxidoreductases. This study provides insights into the structural properties of catalytic redox-active Cys and should further help to recognize thiol oxidoreductases in protein sequence and structure databases.     

Periplasmic protein thiol:disulfide oxidoreductases of Escherichia coli

Disulfide bond formation is part of the folding pathway for many periplasmic and outer membrane proteins that contain structural disulfide bonds. In Escherichia coli, a broad variety of periplasmic protein thiol:disulfide oxidoreductases have been identified in recent years, which substantially contribute to this pathway. Like the well-known cytoplasmic thioredoxins and glutaredoxins, these periplasmic protein thiol:disulfide oxidoreductases contain the conserved C-X-X-C motif in their active site. Most of them have a domain that displays the thioredoxin-like fold. In contrast to the cytoplasmic system, which consists exclusively of reducing proteins, the periplasmic oxidoreductases have either an oxidising, a reducing or an isomerisation activity. Apart from understanding their physiological role, it is of interest to learn how these proteins interact with their target molecules and how they are recycled as electron donors or acceptors. This review reflects the recently made efforts to elucidate the sources of oxidising and reducing power in the periplasm as well as the different properties of certain periplasmic protein thiol:disulfide oxidoreductases of E. coli.