The exact mechanism for the oxidation of succinate to fumarate has not yet been elucidated. In the proposed E1cb mechanism, the deprotonation leads to the formation of an enolate intermediate; FAD then removes the hydride, as shown in Image 2 [9]. Ubiquinone is initially oriented in the active site such that the O1 carbonyl group interacts with Tyr83 of SdhD via hydrogen bonding.
The electrons removed during the oxidation reaction are conveyed through the iron-sulfur clusters to 3Fe-4S; their presence on that cluster stimulates the substrate to reorient so that a second hydrogen bond between the of SdhC may form.
The electrons are transferred to the substrate individually, with the addition of the first producing a radical semiquinone and the second completing the reduction to ubiquinol.
This mechanism is illustrated in image 3 [9]. Since succinate dehydrogenase possesses multiple active sites that catalyze two different reactions, two classes of inhibitors function on the enzyme. The first class, which includes succinate analogs--both naturally-occuring TCA cycle intermediates like malate and oxaloacetate and the synthetic analog, malonate--contains some of the strongest succinate dehydrogenase inhibitors.
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Please note that if you reject them, you may not be able to use all the functionalities of the site. Heart disease, kidney problems, and difficulty breathing can also occur in people with this disorder. The one child died suddenly at the age of five months from a severe deterioration of neuromuscular, cardiac, and hepatic symptoms after an intermittent infection. These genetic changes disrupt the activity of the SDH enzyme, impairing the ability of mitochondria to produce energy.
This suggested a role of additional nuclear genes involved in synthesis, assembly, or maintenance of SDH. It is not known, however, how mutations in the SDHA gene are related to the specific features of Leigh syndrome [ 41 , 42 ]. Two plausible hypotheses have been proposed to explain the peculiar linkage between disruption of electron flow through mitochondrial complex II and tumorigenesis in neuroendocrine cells.
Although certain mutations in these genes result in ROS production in Saccharomyces cerevisiae and mammalian cell lines, it is not clear that ROS accumulate to levels that are mutagenic. ROS model [ 18 ]. Succinate accumulation model [ 18 ]. Excess succinate is shuttled from the mitochondrial matrix to the cytoplasm, where it inhibits any of several aKG-dependent enzymes E that regulate levels or activities of important regulatory proteins black box.
Succinate can then act as an inhibitor of a-ketoglutarate-dependent enzymes that use ferrous iron and molecular oxygen as cofactors to hydroxylate their substrates and generate succinate as a product. It has been demonstrated that two a-ketoglutarate -dependent enzymes, the prolyl hydroxylases, are inhibited by succinate accumulation in cells that have lost SDHD function. Metabolic engineering, i.
In contrast to classical methods of genetic strain improvement such as selection, mutagenesis, mating, and hybridization, metabolic engineering confers two major advantages: 1 the directed modification of strains without the accumulation of unfavorable mutations and 2 the introduction of genes from foreign organisms to equip S.
The latter is particularly crucial for industrial biotechnology to provide pathways that extend the spectrum of usable industrial media e. Since the first introduction of metabolic engineering, there have been tremendous enhancements of its toolbox, and several related disciplines have emerged, such as inverse metabolic engineering and evolutionary engineering. These developments have strongly influenced yeast strain improvement programs in the past few years and have greatly enhanced the potential for using yeast in biotechnological production processes [ 43 ].
The main goals of metabolic engineering can be summarized in the following four categories: 1 improvement of yield, productivity and overall cellular physiology, 2 extension of the substrate range, 3 deletion or reduction of by-product formation and 4 introduction of pathways leading to new products.
Commonly these goals can be achieved by a three-step procedure. Firstly, a genetic modification is proposed, based on metabolic models. After genetic modification, the recombinant strain is analysed and the results are then used to identify the next target for genetic manipulation, if necessary. Thus, the construction of an optimal strain involves a close interaction between synthesis and analysis, usually for several consecutive rounds. The rapid development and frequent success in this field is demonstrated by the large number of reviews about the theoretical and practical aspects of metabolic engineering.
Knowledge of cellular and microbial physiology, as well as the underlying metabolic networks or enzymes, is an important prerequisite for successful engineering. Recently, a computational approach for the identification of every possible biochemical reaction from a given set of enzyme reaction rules was reported.
This analysis suggested that the native pathways are thermodynamically more favorable than the alternative possible pathways. The pathways generated involve compounds that exist in biological databases, as well as compounds that exist in chemical databases and novel compounds, suggesting novel biochemical routes for these compounds and the existence of biochemical compounds that remain to be discovered or synthesized through enzyme and pathway engineering [ 45 ].
Due to its importance in traditional biotechnology such as baking, brewing, and wine making, research activities historically have focused on the yeast Saccharomyces cerevisiae.
It is relatively tolerant to low pH values and high sugar and ethanol concentrations, i. These features are the major reasons for increasing S. Among these compounds, several organic acids may fulfill a role as platform molecules using their multiple functional groups as a target for enzymic or chemical catalysis [ 43 ].
In the United States were identified 10 organic acids as key chemical building blocks [ 44 ]. Similarly, the European focus group BREW identified 21 key compounds that can be produced from different, including renewable sources, a number of which were organic acids [ 45 ]. One example of such a chemical is succinic acid. Succinic acid is used as a surfactant, detergent or foaming agent, as an ion chelator, and also in the food industry as an acidulant, flavoring agent or anti-microbial agent, as well as in health-related products such pharmaceuticals and antibiotics.
Currently, it is produced from petrol and is too expensive to be used as a general building-block chemical. However, provided that its price becomes competitive, succinic acid could replace petrol-derived maleic anhydride in chemical synthesis processes in the future [ 46 - 47 ].
Similar chemical derivatizations can be applied to malic and fumaric acid, so that they can also be considered interesting C 4 building blocks [ 48 - 53 ]. The chemical behavior of the dicarboxylic acid — succinic acid is determined principally by its two carboxyl groups.
This substance is either directly utilized in the pharmaceutical or chemical industry or represent building block or precursor for further chemical or enzymatic syntheses. The following reactions and derivatives are considered interesting: 1 reductions of succinic acid to 1,4-butanediol, -butyrolactone, tetrahydrofuran and its derivatives; 2 reductive amination of succinic acid or -butyrolactone to pyrrolidiones; 3 polymerization of succinic acid with diols building block of polyesters ; 4 polymerisation of succinic acid with diamines to form polyamides, etc.
The examples of the substances that can be derived from succinic acid are shown in table 2. Butanediol, tetrahydrofuran and -butyrolactone, are standard substances for the chemical industry. These are used as solvents, as well as for fiber and polymer production. Dimethylsuccinate is one of the so-called dibasic esters that have great potential as solvents with environmentally benign characteristics.
Thus, the potential market volume for succinic acid is high, fuelling substantial efforts to establish a microbial process for succinic acid production [ 47 ].
The chemical synthesis of succinic acid is predominantly based on maleic anhydride and requires heavy metal catalysts, organic solvents, high temperatures and high pressures. It makes the conversion of maleic anhydride to succinic acid costly and ecologically questionable [ 49 ]. On the other hand, succinate is produced naturally by many microorganisms as an intermediate of the central metabolism or as a fermentation end product.
The succinate producers include bacterial strains, e. Mannheimia succiniproducens [ 54 ]. However, none of these microorganisms are currently used in industry. Some prokaryotes. Examples of various substances that can be derived from succinic acid by chemical conversion [ 47 ]. These restrictions provide strong incentives to integrate and optimize succinate production pathways in other microorganisms via metabolic engineering approaches.
The popularity of S. The yeast S. Saccharomyces cerevisiae grows well in a simple chemically defined medium, under acidic conditions, even at pH values equal 3.
At such low pH values, many weak acids, including succinate, occur predominantly in their undissociated form. This is advantageous for industrial production, as it reduces the need for titration with alkali and allows for direct recovery of undissociated acids. Consequently, there is no need for large quantities of acidifying agents, and the formation of salt byproducts e. In addition, S. The yeast-based fermentation process, which operates at a much lower pH than competing processes, allows succinic acid to be produced with a significantly higher energy efficiency compared to the traditional method.
This compound is not accumulated intracellularly. It is also one of the first bio-based processes that sequesters carbon dioxide in the production process [ 47 , 53 ].
This makes the yeast Saccharomyces cerevisiae a suitable and promising candidate for the biotechnological production of succinic acid on an industrial scale. The metabolic engineering strategy was used for the oxidative production of succinic acid by deletion SDH1, SDH2 genes in the genome. Arikawa et al. In comparison to the wild-type, succinate levels were increased up to2.
The single deletion of gene SDH1 led to a1. Raab et al. The genes SDH1, SDH2, IDH1 and IDP1 , which encode mitochondrial enzymes were deleted with the aim to disrupt succinate and isocitrate dehydrogenase activity to redirect the carbon flux and to allow succinate to accumulate as an end-product.
This study showed that the yeast S. The constructed yeast strains with disruptions in the TCA cycle produced succinic acid up to 3. Saccharomyces cerevisiae is one of the most highly researched model organisms in different biological studies. Using this yeast we can effectively re-examine long-standing and fundamental questions regarding regulation of metabolism and prediction of dynamic models in various cells, including mammalian tissues.
This is a considerable knowledge about the composition, enzymology and membrane binding of the enzyme and relatively new discoveries about its genetics and biosynthesis. Through such efforts, we are able to identify key features of cellular metabolic pathways which can be use both in medicine and in different biotechnological processes. I wish to thank dr Joanna Berlowska from Technical University of Lodz for her help in the making of figures and selection of images of yeast cells.
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