Cofactor NAD H

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. 

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

For the majority of redox enzymes, nicotinamide adenine dinucleotide [NAD(H)j and its respective phosphate [NADP(H)] are required. These cofactors are prohibitively expensive if used in stoichiometric amounts. Since it is only the oxidation state of the cofactor that changes during the reaction, it may be regenerated in situ by using a second redox reaction to allow it to re-enter the reaction cycle. Usually in the heterotrophic organism-catalyzed reduction, formate, glucose, and simple alcohols such as ethanol and 2-propanol are used to transform the... 

Formally, in its oxidized state the cofactor NAD+ is charged negatively due to the two phosphate groups the positive charge denotes quaternization of the nitrogen. It is noteworthy that from the reduced form only the 1,4-NAD(P)H instead of the 1,6-NAD(P)H is enzyme-active, which imposes some restrictions on the regeneration systems in terms of the selectivity. 

Wandrey, C. and Bossow, B. (1986) Continuous cofactor regeneration— utilization of polymer bound NAD(H) for the production of optically active acids. Biotechnol... 

A solution to this problem is the enzyme membrane reactor (Figure 10.8). This is a kind of CSTR (continuous stirred tank reactor), with retains the enzyme and the cofactor using an ultrafiltration membrane. This membrane has a cut-off of about 10000. Enzymes usually have a molecular mass of 25000-250000, but the molecular mass of NAD(H) is much too low for retention. Therefore it is first derivatized with polyethylene glycol (PEG 20000). The reactivity of NAD(H) is hardly affected by the derivatization with this soluble polymer. Alanine can now be produced continuously by high concentrations of both enzymes and of NAD (H) in this reactor. 

Dehydrogenases are very valuable en2ymes in biocatalysis, although one of their challenges is the use of an additional cofactor, such as NAD(P)(H). There are several advantages to dealing only with dehydrogenases that are dependent on NAD(H) but not on NADP(H) ... 

Photosensitized regeneration of an oxidized cofactor, i.e. NAD(P)+ or a reduced cofactor NAD(P)H, could provide general routes for oxidative or reductive biocatalyzed photosynthetic transformations (see Sect. 3.2.3). Coupling of the regenerated NAD(P)+/NAD(P)H cofactors to enzymes that depend on these cofactors, opens a broad array of feasible photo-biocatalytic syntheses. An alternative approach to couple enzymes as catalysts for artificial photosynthetic... 

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. 

What are the bottelnecks for bioreduction The drawbacks of a bioreduction process involving whole cells of microoganisms can be summarized i) Microbial strains possessing both carbonyl reductase activity and cofactor (NAD(P)H)-regenerating activity are necessary to obtain a highmolar yield, because a stoichiometric amount of cofactor is required for substrate reduc-... 

In addition to serving as structural motifs, enols and enolates are involved in diverse biological processes. Several enol/enolate intermediates have been proposed to be involved in glycolysis (Section IV.A), wherein c/ -enediol 21 is proposed to be an intermediate in the catalytic mechanism of phosphohexose isomerase and an enol-containing enamine intermediate (22) has been proposed in the catalytic pathway of class I aldolase. In the case of glucose-fructose (aldose-ketose) isomerization, removal of the proton on Cl-OH produces the aldose while deprotonation of C2-OH yields the ketose, which is accompanied by protonation at the C2 and Cl positions, respectively. There are several cofactors that are involved in various biological reactions, such as NAD(H)/NADP(H) in redox reaction and coenzyme A in group transfer reactions. Pyridoxal phosphate (PLP, 23) is a widely distributed enzyme cofactor involved in the formation of a-keto acids, L/D-amino... 

The application of the same principle to the formation of o-alanine is possible but lacks applicability due to its slowness. In this case, in the presence of ammonia, pyruvate is in equilibrium with its imine. This is reduced at the cathode under formation of racemic alanine. The L-alanine of the racemic mixture is reoxidized by L-alanine dehydrogenase under anodic regeneration of the necessary cofactor NAD to give pyruvate and ammonia, while the o-alanine is not accepted by the enzyme and accumulates in the reaction mixture. The drawback of this reaction is the kinetic control by imine formation, which is very slow, so that a complete inversion of a lOmAf solution of L-alanine would require 140 h [105]. 

Oxidoreductases catalyze oxidation and reduction reactions that occur within the cell. They are very appealing for industrial uses because of the reactions that they are able to catalyze. However, they often need expensive cofactors such as nicotinamide adenine dinucleotides (e.g., NAD+/NADH) and flavines (e.g., FAD/FADH2) in the reactions. In fact, nicotinamide adenine dinucleotides are required by about 80% of oxidoreductases. Fortunately, several NAD(H)... 

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). 

Figure 16. A schematic view of the flow of carbon within the tricarboxylic-acid cycle. The labels m, c, and b designate respectively carbon from the methyl and carboxyl positions of acetyl-CoA and from bicarbonate used to produce oxaloacetate from phosphoenolpymvate. Inputs and outputs of water, of redox cofactors (NAD, etc.), and of coenzymes (CoASH) have been omitted in order to focus on the carbon skeletons. The amination of a-ketoglutarate in order to produce glu is the principal means of importing N for use in the amino-acid pool. The process involves multiple steps, including a reduction (indicated by addition of [H]) and is represented here only schematically. The boldface T indicates transamination, an example of which is shown in equation 21. The circled P represents a phosphate group, POs. Pj represents inorganic phosphate, HP04 .

The hydrogen atoms and their accompanying electrons generated in the TCA oxidation steps captured by the cofactors NAD and FAD now enter a third pathway called the electron transport pathway. Electrons are passed from protein to protein in this pathway in oxidation-reduction steps, and finally are combined with oxygen to form water. Compare these two separate processes for the production of CO2 and H2O with the simultaneous production of the same two substances from the combustion of glucose. The fate of H atoms and their electrons will be discussed in Sec. 22.6. 

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