Nicotinamide cofactors NAD

The most important coenzymes in synthetic organic chemistry [14] and industrially applied biotransformations [15] are the nicotinamide cofactors NAD/ H (3a/8a, Scheme 43.1) and NAD(P)/H (3b/8b, Scheme 43.1). These pyridine nucleotides are essential components of the cell [16]. In all the reactions where they are involved, they serve solely as hydride donors or acceptors. The oxidized and reduced form of the molecules are shown in Scheme 43.1, the redox reaction taking place at the C-4 atom of the nicotinamide moiety. 

Similar to the AAOs, the aaDHs catalyze oxidative deamination, forming an oxoacid and ammonia. However, rather than using enzyme-hound FAD as the oxidant, followed hy O2, these enzymes employ nicotinamide cofactors, NAD or NADP, in free solution (Equation (3)). 

Firstly, let us discuss the example of a thermophilic alcohol dehydrogenase from Bacillus stearothermophilus (bsADH) studied by Kohen et al. [91, 92]. This enzyme catalyzes the abstraction of a hydride to the nicotinamide cofactor NAD+ as depicted in Fig. 6.54. The Arrhenius diagram is depicted in Fig. 6.54(a) a sudden decrease in the apparent slope and the apparent intercept of the Arrhenius curves is observed around room temperature (Fig. 6.54(b)). The puzzling observation is that the kinetic isotope effects are independent of temperature in the high-temper-ature regime but dependent on temperature in the low-temperature regime. 

Regeneration. The oxidized nicotinamide cofactors (NAD(P) ) are considerably more difficult to work with than ATP, but are more tractable than the reduced nicotinamide cofactors (NAD(P)H). The oxidized cofactors are sensitive to nucleophiles (8), but are relatively stable at pH 7 the reduced cofactors decompose by acid-catalyzed processes involving protonation at C-5 of the dihydropyridine ring as the rate-limiting step (equation iii) (9,10). 

Howaldt M, Kulbe KD, Chmiel H (1990) A continuous enzyme membrane reactor retaining native nicotinamide cofactor NAD (H). Ann NY Acad Sci... 

Scheme 43.1 Oxidized (left, NAD(P), 3a/3b) and reduced (right, NAD(P)H, 8a/8b) forms of nicotinamide cofactors.

Until now, only a few versatile, selective and effective transition-metal complexes have been applied in nicotinamide cofactor reduction. The TOFs are well within the same order of magnitude for all systems studied, and are within the same range as reported for the hydrogenase enzyme thus, the catalytic efficiency is comparable. The most versatile complex Cp Rh(bpy) (9) stands out due to its acceptance of NAD+ and NADP+, acceptance of various redox equivalents (formate, hydrogen and electrons), and its high selectivity towards enzymatically active 1,4-NAD(P)H. 

Another approach to preparing enantiomerically pure carboxylic acids and related compounds is via enanhoselective reduction of conjugated double bonds using NAD(P)H-dependent enoate reductases (EREDs EC 1.3.1.X), members of the so-called Old Yellow Enzyme family [44]. EREDs are ubiquitous in nature and their catalytic mechanism is well documented [45]. They contain a catalytic flavin cofactor and a stoichiometric nicotinamide cofactor which must be regenerated (Scheme 6.23). 

Isolated oxidoreductases always depend on cofactors for the transfer of electrons. Enzyme groups which are well characterized with respect to their biochemistry are those requiring the nicotinamide coenzymes NAD or NADP, the flavins FAD or FMN and the ortho-quinoids such as pyrroloquinoline quinone (PQQ) or trihydroxy-phenylalanine (TOPA). 

NMR and ultraviolet (UV) methods have been used to study the interesting adducts formed between the antituberculous drug isoniazid and cofactor NAD (nicotinamide adenine dinucleotide) <20050BC670>. Studies... 

There are two other cofactors that can participate in redox processes these are /lavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADP+). both of which are shown in Fig. 11-2. FAD accepts 2H s and is thereby reduced to FADH2, whereas NADP+ accepts H and is reduced to NADPH and H +. Both of these reduced cofactors can be oxidized, thereby donating their H s (or reducing equivalents), similar to the oxidation of NADH. The enzymes that catalyze those reactions involving an oxidation or a reduction are usually very selective toward a particular cofactor (NAD or NADP) in a particular oxidation state. 

Fig. 2 Different principles for the regeneration of nicotinamide cofactors. Method 1 describes the regeneration using a second enzyme, method 2 shows the substrate-coupled approach utilizing one enzyme for the main reaction, the reduction of the substrate as well as for the regeneration of NAD(P)H...

Direct electrochemical reduction of oxidized nicotinamide cofactors is not useful because of the formation of dimers via intermediate radicals. On the other hand, direct electrochemical oxidation of NAD(P)H to NAD(P)+ can be performed successfully [90]. However, it requires relatively high oxidation potentials and may result in electrode passivation. 

The 3-carbamidopyridinium ring is the chemically active portion of the enzymatic cofactors, NAD and NADP (nicotinamide adenine dinucleotide and its phosphate). A typical reaction involving NAD is the stereospecific (with respect to both cofactor and substrate) oxidation of ethanol to acetaldehyde catalyzed by the enzyme, alcohol dehydrogenase (Eq. 33). 

Assay techniques GDH utilizes both nicotinamide nucleotide cofactors NAD+ in the direction of N liberation (catabolic) and NADP+ for N incorporation (assimilatory). In the forward reaction, GDH catalyzes the synthesis of amino acids from free ammonium and Qt-kg. The reverse reaction links amino acid metabolism with TCA cycle activity. In the reverse reaction, GDH provides an oxidizable carbon source used for the production of energy as weU as a reduced electron carrier, NADH, and production of NH4+. As for other enzymes, spectrophotmetric methods have been developed for measuring oxoglutarate and aminotransferase activities by assaying substrates and products of the GDH catalyzed reaction (Ahmad and Hellebust, 1989). 

All biological C=X CHXH reductions are dependent on the nicotinamide coenzymes NAD(P)H, as illustrated for ketone reduction in equation (1), which shows only the reactive dihydronicotinamide moiety of the NAD(P)H cofactor. 

Figure 1. Stereoview of the active site of ADH looking through the protein and out through the entrance cavity of the receptor site. The zinc atom ( ) is tetra-hedrally coordinated to a his and two cys residues. The forth position is occupied by the O atom of the substrate, here, cyclohexanol. The cofactor, NAD-, is shown from front left to center, where the nicotinamide moiety is placed in bonding proximity with the substrate. 

In Fig. 7 we show results for the heart LDH isoform (1) the Fourier transform of the force on the transferred hydride (left) (2) the Fourier transform of the relative motion between the substrate C2 carbon and carbon C4N of the nicotinamide ring of the cofactor NAD +/NADH (right). In Fig. 8 we show the corresponding figures... 

In Fig. 9 we show the results of a 30-ps simulation for the donor-acceptor distance, i.e. the distance between the C2 carbon of substrate and carbon C4N of the nicotinamide ring of the cofactor NAD + /NADH. Fig. 9 shows that the average donor-acceptor distance is shorter for the heart isoform when lactate and NAD + are bound, and for the muscle isoform when pyruvate and NADH are bound. We propose that the different kinetic activity of the two isoforms is due to the reduced donor-acceptor distance when lactate is bound to the heart isoform and pyruvate is bound to the muscle isoform. 

Enzymes are outstandingly active and highly selective catalysts [332], however, their use in synthesis is often limited by the lack of appropriate cofactors. Reduced nicotinamide cofactors, NADH and NAD(P)H play an important role in many enzyme-catalyzed reactions of practical interest. For example, cyclohexanone and 2-norbomanone was reduced by horse liver alcohol dehydrogenase to afford cyclohexanol, exo-norbomanol (72 %, 38 % e.e.) and enrfo-norbomanol (28 %, 100 % e.e.) on the expense of NADH [333] (Scheme 3.52). 

There are many examples of the use of NAD(P)H to effect organic syntheses in systems in which the reduce d nicotinamide cofactor is regenerated in situ (15,16,24). Recent examples include syntheses of D-lactic acid (10,12), isocitric acid (10,21) and other a-hydroxy acids (D or L) on scales of 0.1 to 0.5 mole (equation ix). The turnover numbers for NAD(P)(H) in these reactions are TTN - 1000 - 2000. 

Synthesis of NAD and NADP . The nicotinamide cofactors are now isolated from yeast (25,26). A major difficulty in this preparation is simply that of separation of the NAD(P)(H) from the other components in the cell. To reduce the cost of these materials, either the yield must be improved from the yeast preparation, the isolation must be simplified, or some type of synthesis must be developed. We have taken a step toward developing a new synthesis by the combined enzymatic/conventional synthetic procedure summarized in Figure 3(27). The overall conversion from... 

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