NAD-malic enzyme

NAD-malic enzyme, hydride transfer from C2 of malate to NAD or APAD, concomitant with decarhoxylation. 

Karsten, W.E., Hwang, C.C. and Cook, P.F. (1999). Alpha-secondary tritium kinetic isotope effects indicate hydrogen tunneling and coupled motion occur in the oxidation of L-malate by NAD-malic enzyme. Biochemistry 38, 4398-4402... 

Karsten, W.E., Gavva, S.R., Park, S.H. and Cook, P.E. (1995). Metal ion activator effects on intrinsic isotope effects for hydride transfer from decarboxylation in the reaction catalyzed by the NAD-malic enzyme from Ascaris suum. Biochemistry 34, 3253-3260... 

NAD-malic enzyme, 52t pentaerithyritol tetranitrate (PETN) reductase, 50t... 

NAD-malic enzyme, 52t, 62-63 A,A-dimethyl-2-(aminomethyl)phenyl benzylselenoxide, 103 A,A-dimethyl-2-(aminomethyl)phenyl bromotelluronium iodide, 91 A,A-dimethyl-2-(aminomethyl)phenyl iodotelluronium iodide, 91 A,A-dimethyl-2-(aminomethyl)phenyl phenyltelluride, 110 Non-covalent molecular structures... 

The C4 cycle can be viewed as an ATP-dependent C02 pump that delivers C02 from the mesophyll cells to the bundle-sheath cells, thereby suppressing photorespiration (Hatch and Osmond, 1976). The development of the C4 syndrome has resulted in considerable modifications of inter- and intracellular transport processes. Perhaps the most striking development with regard to the formation of assimilates is that sucrose and starch formation are not only compartmented within cells, but in C4 plants also may be largely compartmented between mesophyll and bundle-sheath cells. This has been achieved together with a profound alteration of the Benson-Calvin cycle function, in that 3PGA reduction is shared between the bundle-sheath and mesophyll chloroplasts in all the C4 subtypes. Moreover, since C4 plants are polyphyletic in origin, several different metabolic and structural answers have arisen in response to the same problem of how to concentrate C02. C4 plants have three distinct mechanisms based on decarboxylation by NADP+-malic enzyme, by NAD+-malic enzyme, or by phosphoenolpy-ruvate (PEP) carboxykinase in the bundle-sheath (Hatch and Osmond, 1976). 

Two other types of C4 pathways are recognized. In type-2 plants, Atriplex spongiosa) and type-3 Panicum maximum) plants, malate is replaced by aspartate as the major C4 acid transported to the bundle sheath cells. After transport, aspartate is converted to OAA by transamination. In type-2 plants, OAA is reduced to malate, which in turn is decarboxylated by NAD-malic enzyme in the bundle sheath cell mitochondria to give NADH, CO2 and pyruvate. In type-3 plants, OAA is decarboxylated in the cytosol by PEP carboxykinase in the presence of ATP, yielding PEP, CO2 and ADP. The return of carbon to the mesophyll cells for regeneration of the CO2 acceptor occurs as pyruvate (or alanine to maintain nitrogen balance) in type-2 and as PEP (or again perhaps as alanine) in type-3. These variations in the C4 pathway are summarized in Table I (see also Ref. 14). 

A triplex spongiosa NAD-malic enzyme Aspartate Alanine/pyruvate... 

Fig. 3. Carbon flow during Crassulacean acid metabolism (CAM). The simplifled pathway shown is that occurring in malic enzyme type plants. The location of the decarboxylation reaction is believed to be the mitochondria (NAD-malic enzyme type) or the cytosol [16] or chloroplast (NADP-malic enzyme type) [15]. Abbreviations G6P, glucose 6-phosphate F6P, fructose 6-phosphate F16P, fructose 1,6-bisphosphate GAP, glyceraldehyde 3-phosphate PEP, phosphoeno/pyruvate PYR, pyruvate.

These two forms possessed anatomical structures of culms clearly differing from each other. The terrestrial forms had an unusual Kranz type of anatomy which is characterized by the presence of colourless mestome sheath cells intervening between the mesophyll cells and the Kranz cells (1,2,3). The chloroplasts with well-developed grana and many large mitochondria were scattered in the Kranz cells, although the terrestrial forms had biochemical features of the NAD-malic enzyme C4 subtype (2,3). The submersed forms possessed large spherical mesophyll cells and reduced vascular bundles, which are characteristic of submersed aquatic plants (2,4). Kranz cells contained relatively smaller chloroplasts than the Kranz cells of the terrestrial forms. 

In the cytoplasm, citrate lyase splits citrate back into acetyl CoA and oxaloacetate. The oxaloacetate returns to the mitochondria to transport additional acetyl CoA. This process is shown in Figure I-15-I and includes the important malic enzyme. This reaction represents an additional source of cytoplasmic NAD PH in liver and adipose tissue, supplementing that from the HMP shunt. 

Malic enzyme (malate dehydrogenase (decarboxylating), EC 1.1.1.39) catalyzes reversible oxidative decarboxylation of malate to pyruvate. The enzyme uses NAD+ as an electron acceptor, but it is also able to utilize NADP+ with lower affinity (Drmota et al. 1996). With a subunit size of approximately 63 kDa, the Trichomonas hydrogenosomal malic enzyme belongs to the family of large, eukaryotic type of malic enzymes. In contrast, the approximately 40-kDa-subunit malic enzyme, located in the cytosol, belongs... 

In this pathway the electrons for the drug reduction are generated by the oxidative decarboxylation of malate catalyzed by the NAD-dependent malic enzyme (malate dehydrogenase (decarboxylating)). The NADH produced by this reaction is reoxidized by an enzyme with NADH ferredoxin oxidoreductase activity that has been recently identified as a homologue of the NADH dehydrogenase (NDH) module of the mitochondrial respiratory complex I (Hrdy et al. 2004 and see Hrdy et al., this volume). The... 

Fig. 4 Scheme of alternative pathway of metronidazole activation in hydrogenosomes of T. vaginalis the malate pathway. Electrons generated by hydrogenosomal malic enzyme (ME) reduce NAD+ to NADH, NADH dehydrogenase (NDH) recycles NADH and transfers electrons to ferredoxin (Fd) which, in a final step, donates electrons for metronidazole reduction. MTZ metronidazole, R-NO2" metronidazole anion radicals. Experimental conditions as in Fig. 3... 

They stated further that, the new adaptive enzyme catalyzing Reaction 3 appears to be similar to the malic enzyme of pigeon liver, although strictly DPN (instead of TPN)-specific. The coenzyme specificity explains the ready occurrence of Reaction 1. Therefore, the authors showed that exogenous NAD was required for the overall reaction (malic acid -> lactic acid), but because this activity was measured manometrically, they never demonstrated the formation of reduced NAD. Similarly, they did not attempt to show that pyruvic acid was the intermediate between L-malic acid and lactic acid. Instead, the formation of pyruvic acid was inferred from the NAD requirement and because the malic acid dissimilation activity remained constant during purification while the lactate dehydrogenase activity decreased (14). In fact, attempts to show any appreciable amounts of pyruvic acid intermediate failed (22). 

It is not surprising that the pyruvic acid intermediate seemed plausible because in a paper earlier in that same year (23), the authors described a malic enzyme from pigeon liver. This enzyme was shown to form appreciable amounts of pyruvic acid from malic acid, but it was NADP instead of NAD specific. The end product was shown to be pyruvic acid by spectrophotometric assay involving lactate dehydrogenase. 

The most confusing aspect of the pathway proposed by Ochoa and his group now rests with the NAD requirement. In proceeding from L-malic acid to L-lactic acid, there is no net change in oxidation state. Yet in whole cells or cell-free extracts, the malo-lactic fermentation will not proceed in the absence of NAD. Therefore, by the proposed mechanism, one is unable to demonstrate the appearance of reduced cofactor, and the NAD specificity cannot be explained as a redox requirement. However, in the time since this mechanism was proposed, an NAD dependent enzyme (glyceraldehyde-3-phosphate dehydrogenase) has been described which requires NAD in a non-redox capacity (29), and it is possible that the same is true for the enzyme causing the malic acid-lactic acid transformation. 

In Leuconostoc oenos ML 34, we have shown oxaloacetic acid decarboxylation manometrically (6, 7, 8). We were also able to demonstate fluorometrically the enzymatic production of reduced NAD with malic acid as a substrate, but, of course, were unable to do so with oxaloacetic acid since no NADH could be formed from this substrate. It is likely that this oxaloacetic acid decarboxylation activity, as in Lactobacillus plantarum, is distinct from the activity causing the malic-lactic transition. It is also possible that oxaloacetic acid decarboxylation is caused by a malic enzyme. However, there is no verified NAD dependent malic oxidoreductase (decarboxylating) enzyme which does so (12). For example, Macrae (31) isolated a malic enzyme from cauliflower bud mitochondria which showed no activity with oxaloacetic acid. Similarly, Saz (32) isolated a malic enzyme from Ascaris lumbricoides which is also inactive toward oxaloacetic acid. True, the Enzyme Commission (12) lists an enzyme described as L-malate NAD oxidoreductase (decarboxylating) (E.C. 1.1.1.38) which is said to be capable of decarboxylating oxaloacetic acid, but its description dates back to the studies of Ochoa and his group, and we now feel this listing may be improper. 

Ochoa reported that malic enzyme from L. plantarum was NAD and not NADP specific. The malic enzyme of cauliflower bud mitochondria (31) is NAD and NADP specific, with NAD being the preferred cofactor. Both the malo-lactic activity and NADH producing activity of the Leuconostoc oenos system (6,7, 8) was strictly NAD specific. Nicotinamide-adenine dinucleotide phosphate, flavin adenine dinucleotide, and flavin mononucleotide could not substitute in either of these activities. 

Some enzymes contain bound NAD+ which oxidizes a substrate alcohol to facilitate a reaction step and is then regenerated. For example, the malolactic enzyme found in some lactic acid bacteria and also in Ascaris decarboxylates L-malate to lactate (Eq. 15-12). This reaction is similar to those of isocitrate dehydrogenase,110-112 6-phosphogluconate dehydrogenase,113 and the malic enzyme (Eq. 13-45)114 which utilize free NAD+ to first dehydrogenate the substrate to a bound oxoacid whose (3 carbonyl group facilitates decarboxylation. Likewise, the bound NAD+ of the malolactic... 

In view of these problems with Ni2+, Mna+ has been used as a probe for Mg2+ with some success. However, it should be noted that there is a difference in radius which may be manifested in different biochemical behaviour (Mn2+, 0.80 A Mg2+, 0.65 A). Thus Mn2+ has been used to probe the Mg2+ site in pyruvate kinase.95 While the Mg2+-activated enzyme is inhibited by Ca2+ and Li+, the Mn2+-activated enzyme is inhibited by Ca2+ and not by Li+. There are also differences in the catalytic and regulatory properties of the NAD+-specific malic enzyme of E. coli,104 depending upon whether the divalent activator is Mn2+ or Mg2+. It is necessary, therefore, to express a cautionary note. These two cations may act in slightly different ways to bring about a similar final result. In the second example it appears that the metal cofactors stabilize two different conformational states of the enzyme. 

In the trematode F. hepatica as well as in the cestodes H. diminuta and Hymenolepis microstoma, malic enzyme is in fact not NAD but NADP-dependent, producing NADPH during the formation of pyruvate (Tielens... 

Fatty acid biosynthesis (and most biosynthetic reactions) requires NADPH to supply the reducing equivalents. Oxaloacetate is used to generate NADPH for biosynthesis in a two-step sequence. The first step is the malate dehydrogenase reaction found in the TCA cycle. This reaction results in the formation of NAD from NADH (the NADH primarily comes from glycolysis). The malate formed is a substrate for the malic enzyme reaction, which makes pyruvate, CO2, and NADPH. Pyruvate is transported into the mitochondria where pyruvate carboxylase uses ATP energy to regenerate oxaloacetate. 

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