Oxaloacetate reduction

The potential of S. cerevisiae for overproduction of dicarboxylic acids has been demonstrated by Zelle et al. (2008), who constructed a strain that produced 59 g/L of malic acid by engineering of pyruvate carboxylation, oxaloacetate reduction, and malate export. Also the overproduction of succinic acid by S. cerevisiae appeared so successful that the company DSM announced to open a commercial scale (10 kt/ year) bio-based succinic acid plant in 2012 (DSM press release 9 May 2011). 

Xu Q, Li S, Fu YQ, Tai C, Huang H (2010) Two-stage utilization of corn straw by Rhizopus oryzae for fumaric acid production. Bioresour Technol 101 6262-6264 Xu G, Liu L, Chen J (2012) Reconstruction of cytosolic fumaric acid biosynthetic pathways in Saccharomyces cerevisiae. Microb Cell Fact 24 11-24 Zelle RM, de Hulster E, van Winden WA, de Waard P, Dijkema C, Winkler AA, Geertman JM, van Dijken JP, Pronk JT, van Maris AJ (2008) Malic acid production by Saccharomyces cerevisiae engineering of pyruvate carboxylation, oxaloacetate reduction, and malate export. Appl Environ Microbiol 74 2766-2777... 

The TCA cycle can now be completed by converting succinate to oxaloacetate. This latter process represents a net oxidation. The TCA cycle breaks it down into (consecutively) an oxidation step, a hydration reaction, and a second oxidation step. The oxidation steps are accompanied by the reduction of an [FAD] and an NAD. The reduced coenzymes, [FADHg] and NADH, subsequently provide reducing power in the electron transport chain. (We see in Chapter 24 that virtually the same chemical strategy is used in /3-oxidation of fatty acids.)... 

One of these alternate models, postulated by Gunter Wachtershanser, involves an archaic version of the TCA cycle running in the reverse (reductive) direction. Reversal of the TCA cycle results in assimilation of CO9 and fixation of carbon as shown. For each turn of the reversed cycle, two carbons are fixed in the formation of isocitrate and two more are fixed in the reductive transformation of acetyl-CoA to oxaloacetate. Thus, for every succinate that enters the reversed cycle, two succinates are returned, making the cycle highly antocatalytic. Because TCA cycle intermediates are involved in many biosynthetic pathways (see Section 20.13), a reversed TCA cycle would be a bountiful and broad source of metabolic substrates. 

The bioluminescent determinations of ethanol, sorbitol, L-lactate and oxaloacetate have been performed with coupled enzymatic systems involving the specific suitable enzymes (Figure 5). The ethanol, sorbitol and lactate assays involved the enzymatic oxidation of these substrates with the concomitant reduction of NAD+ in NADH, which is in turn reoxidized by the bioluminescence bacterial system. Thus, the assay of these compounds could be performed in a one-step procedure, in the presence of NAD+ in excess. Conversely, the oxaloacetate measurement involved the simultaneous consumption of NADH by malate dehydrogenase and bacterial oxidoreductase and was therefore conducted in two steps. 

Kosicki, G. W., Westheimer, F. H. Oxaloacetate decarboxylase from cod. Mechanism of action and stereoselective reduction of pyruvate by borohydride. Biochemistry 7, 4303—4309 (1968). 

Diacids. The microbial generation of mahc, fumaric, and succinic acid essentially imphes Krebs cycle pathway engineering of biocatalytic organisms to overproduce oxaloacetate as the primary four-carbon diacid that subsequently undergoes reduction and dehydration processes (Scheme 2.9). The use of these four-carbon diacids as intermediate chemicals and the state of their desirable microbial production is briefly outlined. 

The answer is C. Pyruvate kinase deficiency is ruled out by the elevated serum lactate levels. The coma is associated with a fasting hypoglycemia, which is indicative of pyruvate carboxylase deficiency. The elevated citrulline and lysine in the serum are due to a reduction of aspartic acid levels, which are caused by the reduced levels of oxaloacetate, the product of the pymvate carboxylase reaction. 

We have now covered one complete turn of the citric acid cycle (Fig. 16-13). A two-carbon acetyl group entered the cycle by combining with oxaloacetate. Two carbon atoms emerged from the cycle as C02 from the oxidation of isocitrate and a-ketoglutarate. The energy released by these oxidations was conserved in the reduction of three NAD+ and one FAD and the produc-... 

One of the first persons to study the oxidation of organic compounds by animal tissues was T. Thunberg, who between 1911 and 1920 discovered about 40 organic compounds that could be oxidized by animal tissues. Salts of succinate, fumarate, malate, and citrate were oxidized the fastest. Well aware of Knoop s (3 oxidation theory, Thunberg proposed a cyclic mechanism for oxidation of acetate. Two molecules of this two-carbon compound were supposed to condense (with reduction) to succinate, which was then oxidized as in the citric acid cycle to oxaloacetate. The latter was decarboxylated to pyruvate, which was oxidatively decarboxylated to acetate to complete the cycle. One of the reactions essential for this cycle could not be verified experimentally. It is left to the reader to recognize which one. 

Use the plot of AiAQ vs. time to calculate AH/min over the linear portion of the curve. Convert the rate in absorbance terms to activity units. One enzyme unit is the amount of malate dehydrogenase that catalyzes the reduction of 1 micromole of oxaloacetate to L-malate in 1 minute under the described assay conditions. The reduction of 1 micromole of oxaloacetate leads to the oxidation of 1 micromole of NADH therefore, Equation E10.4 may be used to calculate the specific activity of malate dehydrogenase. 

To understand why isocitrate dehydrogenase is so intensely regulated we must consider reactions beyond the TCA cycle, and indeed beyond the mitochondrion (fig. 13.15). Of the two compounds citrate and isocitrate, only citrate is transported across the barrier imposed by the mitochondrial membrane. Citrate that passes from the mitochondrion to the cytosol plays a major role in biosynthesis, both because of its immediate regulatory properties and because of the chain of covalent reactions it initiates. In the cytosol citrate undergoes a cleavage reaction in which acetyl-CoA is produced. The other cleavage product, oxaloacetate, can be utilized directly in various biosynthetic reactions or it can be converted to malate. The malate so formed can be returned to the mitochondrion, or it can be converted in the cytosol to pyruvate, which also results in the reduction of NADP+ to NADPH. The pyruvate is either utilized directly in biosynthetic processes, or like malate, can return to the mitochondrion. 

The question is therefore, what are the principal requirements of an autotrophic carbon-fixation mechanism An organic molecule serves as a C02 acceptor molecule, which becomes carboxylated by a carboxylase enzyme. This C02 acceptor molecule needs to be regenerated in a reductive autocatalytic cycle. The product that can be drained off from such a metabolic cycle should be a central cellular metabolite, from which all cellular building blocks for polymers can be derived examples of such central metabolites are acetyl-CoA, pyruvate, oxaloacetate, 2-oxoghitarate, phosphoe-nolpyruvate, and 3-phosphoglycerate. Importantly, the intermediates should not be toxic to the cell. The irreversible steps of the pathway are driven by ATP hydrolysis, while the reduction steps are driven by low-potential reduced coenzymes. 

Reductive citric acid cycle 5 3 NAD(P)H, 1 unknown donor", 2 ferredoxin 2-Oxoglutarate synthase Isocitrate dehydrogenase6 Pyruvate synthase PEP carboxylase C02 C02 C02 HCOJ Acetyl-CoA, pyruvate, PEP, oxaloacetate, succinyl-CoA, 2-oxoglutarate 2-Oxoglutarate synthase, ATP-citrate lyase... 

The dicarboxylate/4-hydroxybutyrate cycle starts from acetyl-CoA, which is reductively carboxylated to pyruvate. Pyruvate is converted to PEP and then car-boxylated to oxaloacetate. The latter is reduced to succinyl-CoA by the reactions of an incomplete reductive citric acid cycle. Succinyl-CoA is reduced to 4-hydroxybu-tyrate, the subsequent conversion of which into two acetyl-CoA molecules proceeds in the same way as in the 3-hydroxypropionate/4-hydroxybutyrate cycle. The cycle can be divided into part 1 transforming acetyl-CoA, one C02 and one bicarbonate to succinyl-CoA via pyruvate, PEP, and oxaloacetate, and part 2 converting succinyl-CoA via 4-hydroxybutyrate into two molecules of acetyl-CoA. This cycle was shown to function in Igrticoccus hospitalis, an anaerobic autotrophic hyperther-mophilic Archaeum (Desulfurococcales) [40]. Moreover, this pathway functions in Thermoproteus neutrophilus (Thermoproteales), where the reductive citric acid cycle was earlier assumed to operate, but was later disproved (W.H. Ramos-Vera et al., unpublished results). 

The interconversion of o -ketoglutarate to glutamate involves the malate-aspartate shutde. This shuttle translocates a-ketoglutarate from mitochondria into the cytoplasm and then converts it to glutamate by the catalytic action of aspartate aminotransferase (McKenna et al., 2006). As part of the malate-aspartate shuttle, NADH is oxidized during reduction of oxaloacetate to malate. Malate diffuses across the outer mitochondrial membrane (Fig. 1.6). From the intermembrane space, the malate-a-ketoglutarate antiporter in the inner membrane transports malate into the matrix. For every malate molecule entering the matrix compartment, one molecule of... 

Fig. 5.8. Respiratory pathways in Echinococcus spp., sites of ATP synthesis (ox) oxidative, and (red) reductive processes PK, pyruvate kinase OAA, oxaloacetate ME(c), ME(m), malic enzyme (cytosolic) or (mitochondrial) FR, fumarate reductase PDH, pyruvate dehydrogenase complex. (After McManus Bryant, 1986.)...

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