The Electron Carriers NADH and NADPHWhat difference does the phosphate group make that the same one isn't or can't be used for both? Is there a anabollc reason for this separation or is it just coincidental? Why can't the two reaction nadph anabolic reactions The phosphate group in NADPH doesn't affect the redox abilities of the molecule, it is too far away from the part of the molecule involved in the electron transfer. You'll find some more where to get anadrol 50 about this in nadph anabolic reactions 2 of "Molecular Biology of the Cell by Alberts et al. NADPH is primarily produced in the oxidative part of the pentose phosphate pathway.
Nicotinamide adenine dinucleotide phosphate - Wikipedia
Reduced nicotinamide adenine dinucleotide phosphate NADPH is an essential electron donor in all organisms. It provides the reducing power that drives numerous anabolic reactions, including those responsible for the biosynthesis of all major cell components and many products in biotechnology. The efficient synthesis of many of these products, however, is limited by the rate of NADPH regeneration.
Hence, a thorough understanding of the reactions involved in the generation of NADPH is required to increase its turnover through rational strain improvement. Traditionally, the main engineering targets for increasing NADPH availability have included the dehydrogenase reactions of the oxidative pentose phosphate pathway and the isocitrate dehydrogenase step of the tricarboxylic acid TCA cycle.
In the current review, the major canonical and non-canonical reactions involved in the production and regeneration of NADPH in prokaryotes are described, and their key enzymes are discussed. In addition, an overview of how different enzymes have been applied to increase NADPH availability and thereby enhance productivity is provided.
Reduced nicotinamide adenine dinucleotide phosphate NADPH is an essential electron donor in all eukaryotes, bacteria, and archaea. Many natural products of industrial importance are complex secondary metabolites, the production of which often involves NADPH-dependent enzymes. To synthesize such products using purified enzyme systems in vitro would require the addition of huge amounts of NADPH in order to sustain production.
From an industrial point of view, this would be too expensive. NADPH can be regenerated enzymatically by complementing the in vitro system with additional enzymatic reactions or by using substrate-coupled reaction systems. However, reduced productivity compared to systems without in situ regeneration and problems associated with enzyme stability make these options unattractive. Microbial in vivo production systems also provide in situ NADPH regeneration and have several advantages when compared to in vitro systems.
For example, microbes are able to grow on inexpensive renewable feedstocks that provide the organisms with reductant for the regeneration of NADPH.
They also contain numerous pathways, involving stable and highly specific enzymes, thus obviating the need for expensive enzyme purification. In addition, our knowledge of natural metabolic pathways is rapidly advancing, allowing for rational design toward product formation Chemler et al. Therefore, it is not surprising that microbial conversion is the preferred method for the synthesis of a range of products. With the possibility of engineering microbial metabolism to facilitate product formation, it became clear that NADPH availability remains a major hurdle in the efficient generation of many products.
These products range from medicinal compounds Chemler et al. Given its involvement in a multitude of crucial biological functions and its importance in biosynthesis, NADPH is without question an essential molecule. Hence, a key question arises: However, the importance of other NADPH-generating enzymes, such as transhydrogenases, glucose dehydrogenases, and non-phosphorylating glyceraldehyde 3-phosphate dehydrogenase GAPN , is becoming clear, indicating that the traditional view is over-simplistic Sauer et al.
In this review, we describe the major canonical and non-canonical biochemical mechanisms that are involved in the production and regeneration of NADPH in prokaryotes and discuss the key enzymes involved.
NADP H is generally thought of solely as a redox carrier that facilitates the transfer of electrons between two redox couples, a role that does not account for the need for constant resynthesis. The enzymes in prokaryotes are less well-studied, but similar activities and proteins have been found in this domain Mather and Knight, ; Everse et al. Most of these reactions have been characterized in eukaryotes, but the ribosylation reactions also play a role in toxin production by pathogenic bacteria Ziegler, ; Pollak et al.
Both pathways have been reviewed recently Pollak et al. NAD kinase is found in archaea Sakuraba et al. The intracellular parasite appears to lack NADK and hence relies completely on the metabolism of its host cell. According to Kawai and Murata , NADK orthologs can be classified into three types according to their substrate specificity: This type of NADK has been identified in gram-positive bacteria and archaea.
This type has been identified in eukaryotes. The latter type of NADK has been identified in gram-negative bacteria. Hence, many papers about its structure, function, and application are available, including reviews by Kawai and Murata , Shi et al. However, the function of the cofactors is different. The ratios for the electron carriers have been reported, and the values can differ by several orders of magnitude, depending on the organism and growth conditions.
This indicates that the actual redox potential of both redox couples can deviate significantly from the standard potential i.
However, even under these conditions, several reactions appeared to be at equilibrium or even slightly endergonic. However, this is generally not the case Fuhrer and Sauer, In addition, the precise NADPH formation rate depends on fluxes through the generating pathways, which in turn vary with different growth conditions Dauner et al.
Therefore, prokaryotes must have other network-wide biochemical mechanisms that maintain the cellular redox balance Fuhrer and Sauer, The exact mechanisms are not fully understood and are beyond the scope of this review, but papers about the topic are available Singh et al.
The present review provides a general overview of the known major NADPH-generating reactions and discusses the key enzymes involved.
The enzymes that comprise the first group are the oxPPP enzymes glucosephosphate dehydrogenase and 6-phosphogluconate dehydrogenase; isocitrate dehydrogenase of the TCA cycle; malic enzyme; and three enzymes involved in non-canonical NADPH-generating reactions: The enzymes in the second group are transhydrogenases NADH: The ED pathway is mainly present in prokaryotes, although some eukaryotes possess a functional ED pathway as well Fabris et al.
A generally held view is that the ED pathway is less important than the EMP pathway with respect to glucose catabolism. However, Fuhrer et al. Solid and dashed lines indicate single and lumped reactions, respectively.
In addition, the dual cofactor specificity that some G6PDHs show is often observed in vitro under saturated conditions. In general, these isozymes are similar, with the same cofactor preferences, but exceptions exist. Similarly, exceptions among 6PGDH isozymes have been identified. As discussed by Zamboni et al. However, the exact functional roles of these isozymes remains to be determined.
The availability of NADPH is of principal importance for various industrially important classes of products, including amino acids, proteins, antibiotics, organic acids, and high-value metabolites. Both strategies have been applied successfully in various prokaryotes, but reduction in growth is a common side effect Lim et al. However, the opposite effect was observed with an archaeal strain recently developed in our lab: Although both strategies have been applied successfully in various prokaryotes, they are not effective in every organism Poulsen et al.
Overexpressing G6PDH could therefore affect the flux of both pathways simultaneously. However, this is generally not observed.
Cells apparently possess the ability to regulate fluxes to ensure a network-wide balancing of NADPH supply and demand Nicolas et al. The exact effects are strongly dependent on the organism, the genetic background of the parent strain, and the environmental growth conditions Summers et al. Their growth rates are similar to or somewhat lower than those of the parent strain, and they are more sensitive to oxidative stress. In addition, glucose-grown mutants generally display enhanced TCA cycle activity.
In contrast, acetate- or pyruvate-grown cells generally display decreased TCA cycle activity Zhao et al. In contrast, prokaryotes with a functional ED pathway generally respond to 6PGDH knockout by rerouting through the ED pathway and by reversing the direction of the non-oxPPP, slightly increasing the flux through the EMP pathway, and activating malic enzyme Jiao et al. Prokaryotes that lack a functional ED pathway display a similar response, but instead of rerouting the flux through the ED pathway, they increase flux through the EMP pathway Zamboni et al.
The IDH reaction is important for the generation of reducing power. Through the generation of 2-oxoglutarate, the reaction also links nitrogen and carbon metabolism and plays an important role in the cellular defense against oxidative damage and detoxification of ROS Muro-Pastor et al.
IDH activity is important in controlling the metabolic flux between both pathways and is affected by various regulatory factors, such as metal ions Murakami et al. The RTCA cycle is a CO 2 fixation pathway, present in some bacteria and archaea, in which four molecules of CO 2 are fixed to produce one molecule of oxaloacetate Shiba et al.
General overview of the TCA cycle and anaplerotic node. Solid and dashed arrows represent single and lumped enzymatic reactions, respectively. Malic enzyme is able to catalyze the decarboxylation of malate and OAA.
Abbreviations for metabolites normal text and enzymes bold text: Moreover, a survey of sequenced prokaryotic genomes by Zhu et al.
However, although the IDH of H. They therefore proposed that the enzyme should not be categorized as an IDH Aoshima and Igarashi, However, in most cases, this leads to little or no activity Murakami et al. The ability of IDH overexpression to enhance glutamate formation in Corynebacterium glutamicum has also been tested Eikmanns et al. However, overexpression of IDH, even in combination with glutamate dehydrogenase, did not enhance glutamate production.
Disruption of IDH has also been investigated. The exact effects of IDH disruption were dependent on the specific conditions and species used, but some general trends were apparent Mcdermott and Kahn, ; Muro-Pastor and Florencio, ; Eikmanns et al.
First, all IDH mutants investigated were glutamate auxotrophs. Second, although glutamate-containing cultivation media sustained the growth of IDH mutants, their growth was slower than that of the parental strains. Moreover, other significant effects observed in the mutant E. However, with respect to citrate synthase, contradictory results have been obtained. Kabir and Shimizu found upregulation of citrate synthase, but an earlier study of E. Enzymes in the first ME class EC: Enzymes in the second class EC: Enzymes in the third class EC: Moreover, MEs that do not fit the general classification scheme exactly, such as those from the gram-negative bacterium Rhizobium meliloti and the hyperthermophilic archaeon Thermococcus kodakarensis , have been described as well Voegele et al.
Moreover, Morimoto et al. All MEs require a divalent cation as a cofactor. As such, ME is involved in the interconversion of C4 and C3 compounds, which is important for maintaining the levels of TCA cycle intermediates anaplerotic reactions and for growth on C4 and C3 compounds and substrates that enter central metabolism via acetyl-CoA, such as acetate, fatty acids, and ethanol.
Although ME can catalyze C3-carboxylation and C4-decarboxylation reactions, it is generally involved in the latter decarboxylation of malate to pyruvate with the concomitant formation of NADPH Voegele et al. However, examples of prokaryotic MEs without a preference for either Fukuda et al.
To determine the exact contribution of ME to the cellular NADPH pool, the physiological direction and flux through the enzyme needs to be established.