Reductive Carboxylation Of Pyruvate Research Articles

Non‐natural Cofactor and Formate‐Driven Reductive Carboxylation of Pyruvate

AbstractA non‐natural cofactor and formate driven system for reductive carboxylation of pyruvate is presented. A formate dehydrogenase (FDH) mutant, FDH*, that favors a non‐natural redox cofactor, nicotinamide cytosine dinucleotide (NCD), for generation of a dedicated reducing equivalent at the expense of formate were acquired. By coupling FDH* and NCD‐dependent malic enzyme (ME*), the successful utilization of formate is demonstrated as both CO2 source and electron donor for reductive carboxylation of pyruvate with a perfect stoichiometry between formate and malate. When 13C‐isotope‐labeled formate was used in in vitro trials, up to 53 % of malate had labeled carbon atom. Upon expression of FDH* and ME* in the model host E. coli, the engineered strain produced more malate in the presence of formate and NCD. This work provides an alternative and atom‐economic strategy for CO2 fixation where formate is used in lieu of CO2 and offers dedicated reducing power.

Non-natural Cofactor and Formate-Driven Reductive Carboxylation of Pyruvate.

A non-natural cofactor and formate driven system for reductive carboxylation of pyruvate is presented. A formate dehydrogenase (FDH) mutant, FDH*, that favors a non-natural redox cofactor, nicotinamide cytosine dinucleotide (NCD), for generation of a dedicated reducing equivalent at the expense of formate were acquired. By coupling FDH* and NCD-dependent malic enzyme (ME*), the successful utilization of formate is demonstrated as both CO2 source and electron donor for reductive carboxylation of pyruvate with a perfect stoichiometry between formate and malate. When 13 C-isotope-labeled formate was used in in vitro trials, up to 53 % of malate had labeled carbon atom. Upon expression of FDH* and ME* in the model host E. coli, the engineered strain produced more malate in the presence of formate and NCD. This work provides an alternative and atom-economic strategy for CO2 fixation where formate is used in lieu of CO2 and offers dedicated reducing power.

Fumarate and cytosolic pH as modulators of the synthesis or consumption of C4 organic acids through NADP-malic enzyme in Arabidopsis thaliana

Arabidopsis thaliana is a plant species that accumulates high levels of organic acids and uses them as carbon, energy and reducing power sources. Among the enzymes that metabolize these compounds, one of the most important ones is malic enzyme (ME). A. thaliana contains four malic enzymes (NADP-ME 1-4) to catalyze the reversible oxidative decarboxylation of malate in the presence of NADP. NADP-ME2 is the only one located in the cell cytosol of all Arabidopsis organs providing most of the total NADP-ME activity. In the present work, the regulation of this key enzyme by fumarate was investigated by kinetic assays, structural analysis and a site-directed mutagenesis approach. The final effect of this metabolite on NADP-ME2 forward activity not only depends on fumarate and substrate concentrations but also on the pH of the reaction medium. Fumarate produced an increase in NADP-ME2 activity by binding to an allosteric site. However at higher concentrations, fumarate caused a competitive inhibition, excluding the substrate malate from binding to the active site. The characterization of ME2-R115A mutant, which is not activated by fumarate, confirms this hypothesis. In addition, the reverse reaction (reductive carboxylation of pyruvate) is also modulated by fumarate, but in a different way. The results indicate pH-dependence of the fumarate modulation with opposite behavior on the two activities analyzed. Thereby, the coordinated action of fumarate over the direct and reverse reactions would allow a precise and specific modulation of the metabolic flux through this enzyme, leading to the synthesis or degradation of C(4) compounds under certain conditions. Thus, the physiological context might be exerting an accurate control of ME activity in planta, through changes in metabolite and substrate concentrations and cytosolic pH.

Characterization of two members of a novel malic enzyme class

The Gram-negative bacterium Rhizobium meliloti contains two distinct malic enzymes. We report the purification of the two isozymes to homogeneity, and their in vitro characterization. Both enzymes exhibit unusually high subunit molecular weights of about 82 kDa. The NAD(P) + specific malic enzyme [EC 1.1.1.39] exhibits positive co-operativity with respect to malate, but Michaelis-Menten type behavior with respect to the co-factors NAD + or NADP +. The enzyme is subject to substrate inhibition, and shows allosteric regulation by acetyl-CoA, an effect that has so far only been described for some NADP + dependent malic enzymes. Its activity is positively regulated by succinate and fumarate. In contrast to the NAD(P) + specific malic enzyme, the NADP + dependent malic enzyme [EC 1.1.1.40] shows Michaelis-Menten type behavior with respect to malate and NADP +. Apart from product inhibition, the enzyme is not subjected to any regulatory mechanism. Neither reductive carboxylation of pyruvate, nor decarboxylation of oxaloacetate, could be detected for either malic enzyme. Our characterization of the two R. meliloti malic enzymes therefore suggests a number of features uncharacteristic for malic enzymes described so far.

Different regulatory properties of the cytosolic and mitochondrial forms of malic enzyme isolated from human brain

The human brain contains a cytosolic and mitochondrial form of NADP +-dependent malic enzyme. To investigate their possible metabolic roles we compared the regulatory properties of these two iso-enzymes. The mitochondrial malic enzyme exhibited a sigmoid substrate saturation curve at low malate concentration which was shifted to the right at both higher pH values and in the presence of low concentration of Mn 2+ or Mg 2+. Succinate or fumarate increased the activity of the mitochondrial malic enzyme at low malate concentration. Both activators shifted the plot of reaction velocity versus malate concentration to the left, and removed sigmoidicity, but the maximum velocity was unaffected. The activation was associated with a decrease in Hill coefficient from 2.3 to 1.1. The human brain cytosolic malic enzyme displayed a hyperbolic substrate saturation kinetics and no sigmoidicity was detected even at high pH and low malate concentrations. Succinate or fumarate exerted no effect on the enzyme activity. Excess of malate inhibited the oxidative decarboxylation catalysed by cytosolic enzyme at pH 7.0 and below. In contrast, decarboxylation catalysed by mitochondrial malic enzyme, was unaffected by the substrate. These results suggest that under in vivo conditions, cytosolic malic enzyme catalyses both oxidative decarboxylation of malate and reductive carboxylation of pyruvate, whereas the role of mitochondrial enzyme is limited to decarboxylation of malate. One may speculate that in vivo the reaction catalysed by cytosolic malic enzyme supplies dicarboxylic acids (anaplerotic function) for the formation of neurotransmitters, while the mitochondrial enzyme regulates the flux rate via Krebs cycle by disposition of the tricarboxylic acid cycle intermediates (cataplerotic function).

Regulatory Characteristics of the Diphosphopyridine Nucleotide-specific Malic Enzyme of Escherichia coli

Abstract The DPN+-specific malic enzyme (EC 1.1.1.40), which catalyzes the reductive carboxylation of pyruvate with concomitant oxidation of DPNH, has been purified from glucose-grown cells of Escherichia coli. The enzyme is inhibited by ATP (and some other nucleoside triphosphates) and coenzyme A in an allosteric manner. Both of the inhibitors cause noncompetitive inhibition when DPN+ or malate is the varied substrate. In addition, the hyperbolic rate-concentration plots for malate in the absence of the inhibitors become sigmoidal in their presence. Aspartate acts as an activator for the enzyme and in its presence the inhibition caused by CoA is reversed, while the degree of inhibition caused by ATP remains unchanged. From these experiments it is deduced that the inhibitory binding sites for CoA and ATP are different from each other. Since quite low levels of CoA are effective in inhibiting the enzyme it is suggested that the enzyme does not function in vivo unless specific deinhibition is brought about by excess aspartate.

Factors influencing the oxidation of externally added citrate by cardiac mitochondria

Summary It has been demonstrated that citric acid is oxidized only slowly by guinea-pig heart mitochondrial preparations and that this oxidation is unaccompanied by phosphorylation. When citrate is co-oxidized with pyruvate, there is a marked increase in the oxidation rate which is far greater than with either substrate alone. In addition, there is coupled phosphorylation with this oxidation. Under anaerobic conditions the combination of citrate and pyruvate with cardiac mitochondria, resulted in a coupled oxidation—reduction reaction in which α-ketoglutarate, CO2 and malate are formed. It has been proposed that TPNH generated in the isocitrate dehydrogenase ( D s-isocitrate:TPN+ oxidoreductase (decarboxylating), EC 1.1.1.42), reaction could be utilized to form malate by reductive carboxylation of pyruvate, and evidence has been presented that the activity of malate dehydrogenase (decarboxylating) ( L -malate:TPN+ oxidoreductase (decarboxylating), formerly known as malic enzyme, EC 1.1.1.40) of the mitochondria is adequate to account for such a mechanism. α-Ketoglutarate and malate thus formed from isocitrate and pyruvate on the outside of the mitochondria, could be oxidized further by enzyme systems within the particles, and the oxidation of these substrates should be accompanied by phosphorylation.

Biochemistry of Biotin

Publisher Summary This chapter focuses on a host of indirect effects observed in biotin deficiency and offers possible explanations for some of these findings. The discussions primarily deal with the results on certain aspects of biotin in intermediary metabolism. The chapter discusses some of the recent data on the biochemistry of biotin. It also discusses the characterization of five biotin enzymes. The effects of biotin deficiency are felt in very many reactions in the intact organism. Biotin has been implicated to play a role in the deamination of aspartate, serine, and threonine in bacteria in the deamination of serine in animals in the reductive carboxylation of pyruvate by the malic enzyme, in the carboxylation of phosphoenolpyruvate by the phosphoenolpyruvate carboxykinase in carbamylation reactions, in tryptophan metabolism, in purine synthesis, in protein synthesis, and in carbohydrate metabolism. As avidin specifically binds biotin, biotin enzymes do not directly mediate these reactions and the effects observed are indirect results of biotin deficiency.

The intake and utilization of carbon by plant roots from C14-labeled urea Part I. The determination of radioactive carbon of plant materials and a preliminary seedling experiment utilizing C14-labeled urea

Abstract Utilizing radioactive carbon, A. Kursanov, A. Kuzin and co-workers1- 0 recently discovered a new function of the plant root; i.e. COs or carbonate can be absorbed by the plant root, transferred to green tissues and, in the presence of light, is transformed into sugar and protein. Absorption of carbon dioxide by roots is connected with ,β-carboxylation of keto acids and with the movement of dicarboxylic acid to leaves. Pot experiments with four crops where the cu-labeled sodium carbonate was used, showed that the carbon of soil carbonate can be taken up by crop plants only when the pH of the soil showed an alkaline reaction. Field experiments showed that; if carbonic acid was placed in the soil in the form of ammonium or potassium carbonate, the growth of sugar beet increased by 10 to 15 percents. In this new function of plant root, the amount of COs absorbed as well as its pathway to complicated carbon compounds should be discussed. Kursanov et al2,3 showed that the amount of CO2 taken up by roots was considerably large, while Overstreet et al7 obtained comparatively law values with barley root. More recently Poel8 observed a considerably large intake of CO2 by barley root, Cl being distributed in amino acids, amines and carboxylic acids of TCA cycle. Jacobson9 indicated that an unequal absorption of cations and anions by plant roots was reflected in gains or losses of inorganic ions and changes in organic acid content of the roots. Further, the same author10 investigated the relationship between CO2 fixation and malic acid metabolism in plant root, on the basis that among other organic acids particularly malic acid might directly or indirectly be implicated in the ion absorption. Young excised barley roots fixed from 1.18 to 7.02% of the C14Oa in three hours, when it was supplied as NaHC14Oa solution. When excess anion absorption occurred in CaBr2 solution, the lower figures were obtained, and higher figures when excess cation absorption occurred in KH3PO4 solution. The Cl14 accumulated in malate was 68 to 92%. He11,12 also suggested that the mechanism of CO2 fixation may be a reductive carboxylation of pyruvate with the help of malic enzyme, or an oxaloacetic acid carboxylase reduction followed by reduction to malate.