NAD Recycling

O. Miyawaki, K. Nakamura, and T. Yano, Experimental investigation of continuous NAD recycling by conjugated enzymes immobilized in ultrafiltration hollow fiber, J. Chem. Eng. Jpn., 15(3), 224-228 (1982). 

Redox reactions catalyzed by alcohol dehydrogenases (e.g., from horse liver, HLADH) may be performed in organic solvents in both the reduction and oxidation mode, if the recycling system is appropriately modified (Sect. 2.2.1). Reduction of aldehydes/ketones and oxidation of alcohols is effected by NADH- or NAD" -recycling, using ethanol or wobutyraldehyde respectively. 

An alternative way of NAD" recycling makes use of a three-enzyme cascade with molecular oxygen as the ultimate oxidant (Scheme 3.30) [339]. As in the methods described above, all the enzymes and cofactors have to be precipitated together. Thus, NADH which is produced by HLADH-catalyzed oxidation of a secondary alcohol is re-oxidized by diaphorase at the expense of pyrroloquinoline quinone (PQQ) [340]. The reduced form of the latter (PQQH2) is spontaneously oxidized by molecular oxygen producing hydrogen peroxide, which, in mm, is destroyed by catalase. 

Why might it be desirable to coordinately lower the levels of nicotinamide deamidase The 8-fold depression in nicotinamide deamidase activity causes excretion of nicotinamide xthR mutants have shown to be "feeders" for nicotinamide auxotrophs, indicating that these strains continuously excrete nicotinamide into the medium. This is presumably a consequence of the pyridine nucleotide cycle, shown in Fig. 2. Nicotinamide deamidase is not only an enzyme for the salvage of exogenous pyridine, but it is part of a NAD recycling pathway, i.e., a "pyridine nucleotide cycle"... 

Figure 7.3 NAD recycling. Humans have two metabolic pathways that are able to recycle nicotinamide. NAD-consuming enzymes (ARTs, PARPs, sirtuins) break down NAD to nicotinamide and ADP-ribosyl product. Nicotinamide by the enzymatic action of nicotinamide phosphoribosyltransferase (NAMP/PBEF) and nicotinamide/nicotinate-mononucleotide-adenyltransferases isoenzymes (NMATl-3) is then retransformed to NAD. In a second pathway, nicotinamide riboside is phosphorylated by nicotinamide riboside kinase (NRK 1,2) to nicotinamide mononucleotide. Subsequently, nicotinamide mononucleotide is converted to NAD by the catalytic action of NMNATs.

The coupling of an oxidation and a reduction stage through NAD recycles the cofactor, just as does Saccharomyces cerevisiae in the synthesis of ethanol (see section 6.2.1.1). Without such coupling each half of the reaction would be limited by the concentration of NAD and this is a general problem which must be faced in the development of many synthetic reactions. 

The second electron shuttle system, called the malate-aspartate shuttle, is shown in Figure 21.34. Oxaloacetate is reduced in the cytosol, acquiring the electrons of NADH (which is oxidized to NAD ). Malate is transported across the inner membrane, where it is reoxidized by malate dehydrogenase, converting NAD to NADH in the matrix. This mitochondrial NADH readily enters the electron transport chain. The oxaloacetate produced in this reaction cannot cross the inner membrane and must be transaminated to form aspartate, which can be transported across the membrane to the cytosolic side. Transamination in the cytosol recycles aspartate back to oxaloacetate. In contrast to the glycerol phosphate shuttle, the malate-aspartate cycle is reversible, and it operates as shown in Figure 21.34 only if the NADH/NAD ratio in the cytosol is higher than the ratio in the matrix. Because this shuttle produces NADH in the matrix, the full 2.5 ATPs per NADH are recovered. 

Many procedures have been suggested to achieve efficient cofactor recycling, including enzymatic and non-enzymatic methods. However, the practical problems associated with the commercial application of coenzyme dependent biocatalysts have not yet been generally solved. Figure A8.18 illustrates the continuous production of L-amino adds in a multi-enzyme-membrane-reactor, where the enzymes together with NAD covalently bound to water soluble polyethylene glycol 20,000 (PEG-20,000-NAD) are retained by means of an ultrafiltration membrane. 

The asymmetric reduction of prochiral functional groups is an extremely useful transformation in organic synthesis. There is an important difference between isolated enzyme-catalyzed reduction reactions and whole cell-catalyzed transformations in terms of the recycling of the essential nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] cofactor. For isolated enzyme-catalyzed reductions, a cofactor recycling system must be introduced to allow the addition of only a catalytic amount (5% mol) of NAD(P)H. For whole cell-catalyzed reductions, cofactor recycling is automatically achieved by the cell, and the addition of a cofactor to the reaction system is normally not required. 

It is possible to use isolated, partially purified enzymes (dehydrogenases) for the reduction of ketones to optically active secondary alcohols. However, a different set of complications arises. The new C H bond is formed by delivery of the hydrogen atom from an enzyme cofactor, nicotinamide adenine dinucleotide (phosphate) NAD(P) in its reduced form. The cofactor is too expensive to be used in a stoichiometric quantity and must be recycled in situ. Recycling methods are relatively simple, using a sacrificial alcohol, or a second enzyme (formate dehydrogenase is popular) but the real and apparent complexity of the ensuing process (Scheme 8)[331 provides too much of a disincentive to investigation by non-experts. 

Biocatalytic approaches to cofactor regeneration can be divided into coupled-enzyme methods and coupled-substrate methods.In the coupled-enzyme method, the oxidized cofactors (NAD+ and NADP+) are recycled in situ by performing an oxidation reaction using a second enzyme and an inexpensive auxiliary substrate. This second enzyme must employ the same cofactor, but neither enzyme should be able to accept the same substrate. 

In the hyperthermophilic Archaea, NAD(P)-reactive enzymes are involved in recycling the reduced cofactors to produce H2 as a waste product as in the case of the NADPH oxidising hydrogenases from the hyperthermophilic Archaea, e.g. Pyrococcus species (Bryant and Adams 1989 Pedroni et al. 1995) and Thermococcus litoralis (Rakhely et al. 1999). These enzymes are also heterotetramers (Fig. 2.2C) with an apparently similar organisation of subunits and prosthetic groups to the Eubacterial examples of Group 5. 

In a similar exercise with D-methionine, Findrik and Vasic-Racki used the D-AAO of Arthrobacter, and for the second-step conversion of oxoacid into L-amino acid, used L-phenylalanine dehydrogenase (L-PheDH), which has a sufficiently broad specificity to accept L-methionine and its corresponding oxoacid as substrates. Efficient quantitative conversion in this latter reaction requires recycling of the cofactor NAD into NADH, and for this the commercially available formate dehydrogenase (FDH) was used (Scheme 2). 

This use of a weaker oxidant has several consequences. First, the reaction is readily reversible. Indeed, at neutral pH and with average substrate concentrations, the equilibrium tends to lie toward amino acid formation. Second, since the oxidant is not an ubiquitous oxygen, with a discardable product, but costly NAD(P)", forming NADPH, it becomes essential in any production process to find a way to reclaim or recycle the cofactor. Third, the absence of H2O2 among the products largely removes the concern about further reaction of the oxoacid through oxidative decarboxylation. 

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