NADP-malate dehydrogenase

A reversible covalent modification that plants use extensively is the reduction of cystine disulfide bridges to sulf-hydryls. Many of the enzymes of photosynthetic carbohydrate synthesis are activated in this way (table 9.3). Some of the enzymes of carbohydrate breakdown are inactivated by the same mechanism. The reductant is a small protein called thioredoxin, which undergoes a complementary oxidation of cysteine residues to cystine (fig. 9.5). Thioredoxin itself is reduced by electron-transfer reactions driven by sunlight, which serves as a signal to switch carbohydrate metabolism from carbohydrate breakdown to synthesis. In one of the regulated enzymes, phosphoribulokinase, one of the freed cysteines probably forms part of the catalytic active site. In nicotinamide-adenine dinucleotide phosphate (NADP)-malate dehydrogenase and fructose-1,6-bis-... 

Fructose-1,6-bisphosphatase Sedoheptulose-1,7-bisphosphatase Glyceraldehyde-3-phosphate dehydrogenase NADP-malate dehydrogenase Phosphoribulokinase Thylakoid ATP-synthase... 

Aside from PEPCase, a number of other CAM-related genes have been partially characterised (Table 1). These include cDNA clones for pyruvate, orthophosphate dikinase (PPDK), a specific NADP malate dehydrogenase (MDH), glyceraldehyde phosphate dehydrogenase (GaPDH) and NADP-dependent malic enzyme (MOD). Previous studies indicated that the enzymatic activities of these gene products increased upon salt stress in the ice plant (Holtum Winter, 1982). As in the case... 

Subsequent metabolism of oxaloacetate (OAA) varies according to species. Three main types of C4 pathway are recognized, of which the most extensively studied is that shown by plants such as Zea mays (corn) (Fig. 2). In these plants (here called type-1 C4 plants) OAA is reduced to malate via NADP-malate dehydrogenase in mesophyll chloroplasts. Malate is then transported to bundle sheath chloroplasts and oxidatively decarboxylated by NADP-malic enzyme to produce pyruvate, CO2 and NADPH. Pyruvate is recycled to the mesophyll cells while the CO2 and NADPH are used in the RPP cycle in the bundle sheath chloroplast. The original C3 carbon acceptor (PEP) is regenerated from pyruvate in the mesophyll chloroplast by the activity of pyruvate, Pj dikinase [8] (Eq. 5). 

It is likely that the regulatory mechanisms discussed in Section 5 apply to the regulation of CO2 fixation in C4 plants. In particular it is known that the ferre-doxin/thioredoxin system of light-linked enzyme activation (see Section 5.2.2) is present in C4 plants. NADP-malate dehydrogenase, FBPase and SBPase from maize leaves are regulated in this way [33]. 

Glucose 6-phosphate dehydrogenase Phenylalanine ammonia lyase Rihulose 5 -phosphate kinase NADP+-malate dehydrogenase... 

Corn leaf NADP- malate dehydrogenase (NADP- MDH), spinach ferredoxin, ferredoxin thioredoxin reductase and thioredoxin m were purified to homogeneity as described earlier (1). Pig ad enal ferredoxin was prepared as in (1). Human lymphocyte thioredoxin was cloned, overexpressed and purified from E coli as described in (2). 

E. coli thioredoxin reductase was purified on 2, 5 -ADP Sepharose (4). E. coli ribonucleotide reductase was a generous gift of B-M. Sjoberg, Stockholm. Ribonucleotide reductase from Anabaena 7119 was purified as previously described (6). NADP-malate dehydrogenase and fructose-1,6-bisphosphatse were partially purified from spinach chloroplasts (7). Thioredoxins m and f were homogeneous preparations from spinach chloroplasts (8). 

Reduced Anabaena T-1 will activate spinach chloroplast NADP-malate dehydrogenase after pre-incubation for a minimum of 10 min (7). Approximately 0.1 jjq/ /jg MDH produced a 2-3 fold stimulation of activity. E. coli thioredoxin was equally effective in this system. Reduced T-2 was completely ineffective. In this respect, it resembles spinach thioredoxin f. 

REGULATION OF NADP-MALATE DEHYDROGENASE LIGHT-ACTIVATION BY THE REDUCING POWER. I FUNCTIONAL STUDIES. 

Figure 2. Effect of changes in irradiance and photoinhibition on the ATP content (A) and NADP-malate dehydrogenase (i) and fructose -1,6-bisphos-phatase (ii) activities (B) of barley leaves.

In chloroplasts oxalacetic acid could be reduceo to malic acid (by NADP-malate dehydrogenase) and this acid could be decarboxylated and will regenerate the substrate for PEPCase. Another reaction is also possible - oxalacetic acid to be directly decarboxylated by oxalacetate decarboxylase. In either case the decarboxylase reactions will strengthen the flow of CO2 to RuBPCase and will contribute to the better operation of Calvin cycle. 

Regulation of NADP-Malate Dehydrogenase Light-Activation by the Reducing Power. 

In addition, succulent plants have a chloroplast-specific NADP malate dehydrogenase analogous to the NADP form reported in C3 and C4 plants (Hatch and... 

Because of the fairly conclusive evidence that malate synthesized by dark CO2 fixation is not in equilibrium with malate synthesized in the mitochondria, it seems reasonable to conclude that the soluble malate dehydrogenase functions to a large extent in the dark CO2 fixation pathway. We also would predict, that insofar as P-enolpyruvate carboxylase-mediated synthesis of malate takes place in chloroplasts, the NADP malate dehydrogenase would be the coupling enzyme. 

NADP-malate dehydrogenase in green leaves is regulated in vivo in a manner similar to the other key mesophyll chloroplast enzyme, pyruvate phosphate-dikinase, i.e. it is rapidly inactivated in the dark and reactivated upon illumination." Maize plants kept in darkness... 

It can be assumed that pyruvate phosphate-dikinase and NADP-malate dehydrogenase have no useful function in the dark and may even have detrimental effects on the metabolic status quo of chloroplasts if they use the ATP or reducing power available. 

Malic acid is mainly produced as an acidulant and taste enhancer in the beverage and food industries. The most preferred metabolic pathway for malate production starts from glucose and proceeds with the carboxylation of pyruvate, followed by the reduction of oxaloacetate to malate. These pathways have been identified in bacterial, yeast, and fungal species (Werpy et al., 2004). In the microalgae reduction of oxaloacetate to malate by NADP malate dehydrogenase (Ouyang et al., 2013 Kuo et al., 2013), the condensation of oxaloacetate and acetyl-coenzyme A (acetyl-CoA) to citric acid is followed by oxidation steps of the tricarboxylic acid (TC A) cycle or glyoxyl-ate shunt to malate (Steinhauser et al., 2012 Pearce et al., 1969 Woodward and Merrett, 1975). 

Henderson, N. S., 1966, Isozymes and genetic control of NADP-malate dehydrogenase in mice. Arch. Biochem. Biophys. 117 28. 

Figure 6. Fatty acid synthesis in mammalian tissues showing the transhydrogenation cycle. Pyruvate is generated from glucose in the cytosol (upper portion of figure) and converted to fatty acids by a reaction sequence involving enzymes in the mitochondrial matrix (lower portion of figure). 1, pyruvate carboxylase 2, ATP citrate lyase 3, NADP-malate dehydrogenase. These reactions were absent in adipose tissue from ruminant animals (Hanson and Ballard, 1967).

We have already described the purification of a chloroplastic NADP-malate dehydrogenase (Ferte et al., 1982). It is a 56 000 daltons dimeric protein made up of two apparently identical subunits. This enzyme plays an important role in the generation of reducing power used in the reduction cytoplasmic reactions. NADPH from photosynthesis reduces oxaloacetate to malate in the stroma. This reaction is catalyzed by NADP-MDH. A malate translocator exports this acid outside the chloroplast where NAD-MDH generates NADH. 

Ferte, N., Meunier, J.C., Ricard, J., Buc, J. and Sauve, P. (1982) Molecular properties and thioredoxin-mediated activation of spinach chloroplastic NADP-malate dehydrogenase. FEBS Letters, 146, 133-138. 

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