NAD-dependent malic enzyme

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... 

Another overexpression strategy was tried with the NAD -dependent malic enzyme of E. coli Thermodynamically, the reduction of pyruvate to malate is favored, but in nature this reaction does not occur. A double mutant of E. coli, NZNlll, which is blocked in both pyruvate formate lyase pjT) and lactate dehydrogenase (Idh), was used as the host. It is unable to grow anaerobically because its pyruvate metabolism is blocked by the fermentation end products acetate, formate, ethanol, and lactic acid. The mutant NZNl 11 with multiple copies of malic enzyme accumulated succinic acid as a major end product only when the cells were switched to anaerobic metabolism gradually by metabolic depletion of oxygen in a sealed tube (Clark et al. 1988). Mutant strains blocked in either pfl or Idh did not alter their distribution of fermentation products when overexpressing malic enzyme. 

SteinbUchel A, Schmack G (1995) Large-scale production of poly(3-hydroxyvaleric acid) by fermentation of Chromobacterium violaceum, processing, and characterization of the homopolyester. J Environ Polym Degrad 3 243-258 Stols L, Donnelly MI (1997) Production of succinic acid through overexpression of NAD -dependent malic enzyme in an Escherichia coli mutant. Appl Env Microbiol 63 2695-2701... 

Carbon for FAS could also be produced via ATP-citrate lyase. This activity, which will convert citrate to oxaloacetate plus acetyl-CoA, has been demonstrated in soybean extracts. But there is no current evidence for citrate transport into the plastid nor of localisation of this activity in the plctstid to support citrate cleaving enzyme as a source of carbon for FAS, at least for avocado mesoccirp plastids. Extracts of developing soybean also contain a NADP+-dependent medic enzyme.20 NAD+-dependent malic enzyme, which produces pyruvate ind Cctrbon dioxide from malate, is an enzyme specific to the mitochondrial matrix in higher pleints.21 The localisation of NADP+-malic enzyme in immature soybeans, eind the possibility of pyruvate production other than by pyruvate kinase, and the utilisation of this pyruvate in FAS, remain to be determined. 

Diastereoselective synthesis of cyc/o-saligenyl nucleoside phosphotriesters has been achieved using two diastereopure 3-methyl-cyc/o-Sal-phospho-triesters, e.g. (1, nucleoside phosphotriester derivatives of AZT and d4T were prepared and it was found that the antiviral activity was between five and 20-fold different between the individual diastereoisomers. A series of C5-substituted alkenyl- alkynyl- and aryl-dUMP derivatives was synthesised and assayed against the flavin-dependent thymidylate synthase ThyX from Mycobacterium tuberculosis. Of these some were found to be active at submicromolar concentration with specificity for ThyX compared with a bacterial thymidylate synthase. Eleven triazole nucleoside monophosphates (2) were synthesised, with inhibitory effects against bacterial NAD-dependent malic enzyme. ... 

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. 

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. 

Sinorhizobium meliloti could accumulate PHB during fiee life, but not in symbiosis (Figure 1). It was hypothesised that this could be due to a low activity of the NADH-dependent malic enzyme . S. meliloti has two malic enzymes, one NADH dependent (Dme) and the other NADPH dependent (Tme). While Dme and Tme are both expressed in the fiee-living state, Tme is repressed in bacteroids and Dme is inhibited by excess of acetyl-CoA. As a consequence PHB is not accumulated in the bacteroid because die levels of NAD(P)H are too low. 

Driscoll B. and Finan T.M., 1997, Properties of NAD - and NADP -dependent malic enzymes of Rhizobium Sinorhizobium) meliloti and differential expression of their genes in nitrogen-fixing bacteroids. Microbiology 143 489-498. 

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 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... 

It has been purified (445) and shares some properties in common with malic enzymes from mammals and birds in being NADP-dependent, heat-stable and able to decarboxylate oxaloacetate. The malic enzyme of H. microstoma also has a marked specificity for NADP (216), contrasting with that of Spirometra mansonoides, which appears to be both NAD- and NADP-linked (220). Malic enzyme has been demonstrated in a range of other cestodes including Mesocestoides corti (399), Schistocephalus solidus (406), Moniezia expansa (60), Echinococcus spp. (500) and L. intestinalis (502). 

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). 

MDase (L-malate NAD+ oxidoreductase EC 1.1.1.37), used in homogeneous EIA, is prepared from pig heart (mitochondria). The oxidation of L-malate is generally catalyzed by two distinct pyridine nucleotide-dependent enzymes those of the malate-oxaloacetate class, which use NAD+, and those of the malate-pyruvate class (commonly known as malic enzymes), which use NADP+ (Banaszak and Bradshaw, 1975). 

The oxidation of L-malate in most living organisms is catalyzed by two distinct types of pyridine nucleotide-dependent enzymes. In one case the principal product is oxaloacetate, while in the other it is pyruvate and CO2. The enzymes of the malate-oxaloacetate class, which utilize NAD+, have been referred to as simple dehydrogenases, while enzymes of the malate-pyruvate type, which, in contrast, use NADP+, have been designated decarboxylating dehydrogenases and are commonly known as malic enzymes (1). 

FIG. 5.4 Alanine synthesis and the reoxidation of glycolytic NADH. 1, Glycolysis 2, alanine aminotransferase 3, NAD-dependent glutamate dehydrogenase 4, NADP-dependent glutamate dehydrogenase 5, malate dehydrogenase 6, malic enzyme (cytosolic). 

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