NADH/NADPH cofactors

Oxidoreductases these enzymes catalyze redox reactions. Examples are oxidases that catalyze oxidation of a substrate by reducing molecular oxygen (02), and peroxidases that reduce H202. Laccases (EC 1.10.3.2) are oxidases that catalyze the oxidation of (poly)phenolic substrates. Reductases and dehydrogenases (EC 1.1.1) catalyze the reduction of carbonyls, using NADH/NADPH cofactors. Catalases (EC 1.11.1.6) catalyze the decomposition of H202 to 02 and H20. 

Oxidoreductases are a family of enzymes that catalyze a number of industrially important reactions, but they often require additional nicotinamide (NADH or NADPH) cofactors which... 

Ketoreductases catalyze the reversible reduction of ketones and oxidation of alcohols using cofactor NADH/NADPH as the reductant or NAD + /NADP+ as oxidant. Alcohol oxidases catalyze the oxidation of alcohols with dioxygen as the oxidant. Both categories of enzymes belong to the oxidoreductase family. In this chapter, the recent advances in the synthetic application of these two categories of enzymes are described. 

Reduced flavins (FADH2, FMNH2, and riboflavin) generated by flavin-dependent reductases have been hypothesized to reduce azo dyes in a nonspecific chemical reaction, and flavin reductases have been revealed to be indeed anaerobic azoreductases. Other reduced enzyme cofactors, for example, NADH, NADH, NADPH, and an NADPH-generating system, have also been reported to reduce azo dyes. Except for enzyme cofactors, different artificial redox mediating compounds, especially such as quinines, are important redox mediators of azo dye anaerobic reduction (Table 1). 

Relative to the dithiol DTT but also to other monothiols such as 2-mercaptoeth-anol, GSH is a poor stimulator of microsomal deiodinase activity even when tested in the presence of NADPH and glutathione reductase [52,60,61]. Deiodinase activity of isolated microsomes is supported to a limited extent by GSH if tested with low (nM) but not high (/zM) rT3 concentrations or with T4 as the substrate. This low potency of GSH has led investigators to explore other physiological cofactors. As mentioned above, the paucity of cytoplasmic dihydrolipoamide makes it an unlikely candidate despite its unsurpassed potency [52], This is supported by the finding that addition of NADH, the cofactor for lipoamide hydrogenase, does not stimulate deiodinase activity of kidney homogenates unless supplemented with lipoamide [52]. 

Heterofermentative LAB have the capability to utilize high concentrations of fructose such that the mannitol concentration in the fermentation broth could reach more than 180g/L, which is enough to be separated from the cell-free fermentation broth by cooling crystallization. Lactic and acetic acids can be recovered by electrodialysis (Soetaert et al., 1995). The enzyme mannitol dehydrogenase responsible for catalyzing the conversion of fructose to mannitol requires NADPH (NADH) as cofactor. Thus, it is possible to develop a one-pot enzymatic process for production of mannitol from fructose if a cost-effective cofactor regeneration system can be developed (Saha, 2004). The heterofermentative LAB cells can be immobilized in a suitable support, and... 

Enzymatic cofactors, such as nicotinamide adenine dinucleotide (NADH), nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (EAD), flavin mononucleotide (EMN), and pyridoxal phosphate, are fluorescent and commonly found associated with various proteins where they are responsible for electron transport (see Fig. lb and Table 1). NADH and NADPH in the oxidized form are nonfluorescent, whereas conversely the flavins, FAD and EMN, are fluorescent only in the oxidized form. Both NADH and FAD fluorescence is quenched by the adenine found within their cofactor structures, whereas NADH-based cofactors generally remain fluorescent when interacting with protein structures. The fluorescence of these cofactors is often used to study the cofactors interaction with proteins as well as with related enzymatic kinetics (1, 9-12). However, their complex fluorescent characteristics have not led to widespread applications beyond their own intrinsic function. 

But the application of redox enzymes is also connected with intrinsic problems. The difficulty with redox enzymes in synthetic processes is based on the fact that this class of biocatalysts is dependent on freely dissociated (like NADH, NADPH, pteridin) or enzyme-bound (like FMN, FAD, thio-tryosine, PQQ, or cytochrome) cofactors, respectively, prosthetic groups (Fig. 1) in stoichiometric amounts to shuttle the redox equivalents from the enzyme to the substrate. 

Figure 2.8 compares corrected Nernst plots for C-lobe half-transferrin free in solution and bound to the transferrin receptor, at endosomal pH. These data clearly demonstrate that docking iron-loaded C-lobe transferrin at the transferrin receptor at pH 5.8 makes it energetically more favourable to reduce Fe " to Fe by 200 mV. Furthermore, receptor-docking places Fe reduction in a range accessible to NADH or NADPH cofactors, consistent with the hypothesis that reduction is the initial event in iron release from transferrin in the endosome. Fe " is bound by /zTf at least 14 orders of magnitude more weakly than Fe, so that reductive release of iron bound to HTi in the transferrin-transferrin receptor complex is then physiologically and thermodynamically feasible, and the barrier to transport across the endosomal membrane is lifted. The transferrin receptor, therefore, is more than a simple conveyor of... 

Chromate was reduced to chromium(III) by rat liver microsomes and NADPH in vitro ). Reduction of hexavalent chromium to trivalent chromium required the presence of both microsomal protein and NADPH cofactor. Heat denaturation of microsomes resulted in the loss of their ability to reduce chromate. Essentially no chromate reduction was observed by the cofactor in the absence of microsomal proteins. We have found that NADH also served as a cofactor for the microsomal reduction of chromate. However, at equivalent concentrations of NADPH and NADH, the rate of reduction of limiting amounts of the substrate chromate was slower with NADH than with NADPH cofactor. 

The NADPH- and NADH-Cytochrome P-450 reductase activities of microsomes have been shown to be inhibited by 3-pyridinealdehyde-NAD The rate of reduction of chromate by microsomes using NADH cofactor was significantly decreased by the presence of 3-pyridinealdehyde-NAD with NADPH cofactor the rate was substantially slower at equivalent concentrations of inhibitor and cofactor. 

In the oxidation process of an alcohol by NAD+ (or NADP+), the enzymatic base positioned above the carbonyl takes back its proton, and the electrons in the O—H bond shift down and push out the hydride, which is immediately accepted by C-4 of NAD+ (or NADP+). The products are just what they were started out with, a ketone and NADH (or NADPH) (Figure 1.39). The mechanism of oxidation of amines to imines as well as of aldehyde to carboxylate is similar to the oxidation of alcohol. In the reduction process of a ketone by NADH (or NADPH), both the ketone substrate and cofactor are bound in the enzyme s active site, and C-4 of the nicotinamide ring is positioned very close to the carbonyl carbon of the ketone. As an enzymatic group transfers a proton to the ketone oxygen, the carbonyl carbon becomes more electrophilic and is attacked by a hydride from NADH (or NADPH). The ketone is reduced to an alcohol, and the NADH or NADPH cofactor is oxidized to NAD+ or... 

Alternatively, electrochemical detection by using an amperometric biosensor has been proposed using modified electrodes for the electrocatalytic oxidation of the reduced cofactors (NADH, NADPH). The oxidation current reflects the rate of glucose conversion. Additionally, covalent coupling of the coenzyme is a precondition of more advanced reagentless measuring devices. Further developments use an electron mediator such as ferrocyanide and PQQ/ PQQHi (pyrroloquinoline quinone) as the cofactor pair. 

The first indication of the existence of so-called Baeyer-Villiger monooxygenases (BVMOs) was reported in the late 1940s [56]. It was observed that certain fungi were able to oxidize steroids via a BV reaction [56], but two decades elapsed before the first BVMOs were isolated and characterized [57, 58]. All characterized BVMOs contain a flavin cofactor that is vital for the catalytic activity of the enzyme, Furthermore, NADH or NADPH cofactors are needed as electron donors. Careful inspection of all available biochemical data on BVMOs has revealed that at least two discrete classes of BVMOs exist, types I and II [59]. 

Penrose 1978). More recent studies have shown that Mytilus edulis has the capacity to metabolize PAHs and aromatic amines (Marsh et al. 1992) in addition to its ability to metabolize benzo[a]pyrene in the absence of NADH or NADPH cofactors (Lemaire et al. 1993). It has also been demonstrated that a Mytilus sp. can form reactive metabolites of benzo[a]pyrene that attach to DNA (Marsh et al. 1992). 

The stereospecific introduction of a Z-double bond (Kcurs by the abstraction of two vicinal pro-/ hydrogens atoms at C-9 and C-10 in a thioester (Fig. 3.3). In plants, R = ACP, while in animals and fungi, the thioester is activated as CoA. This reaction is catalyzed by stearoyl-ACP A -desaturase in plants and stearoyl-CoA A -desaturase in animals and fungi [1]. Oxygen and either NADPH or NADH as cofactors are required for both types of desaturases. 

Bacteria use two redox carriers nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH). The abbreviations in the parentheses denote the carriers when they are in the reduced form. When these molecules are in their oxidized forms (NAD+, NADP ), they abstract electrons from molecules in the form of a hydride and in the process, become reduced (NADH, NADPH). For example, the reaction A + NAD+ —> B + NADH occurs where B is more oxidized than A, and the electrons are transferred by adding a hydride to NAD to yield the reduced cofactor, NADH. 

All characterized BVMOs contain a flavin cofactor that is crucial for catalysis while NADH or NADPH is needed as electron donor. An interesting observation is the fact that most reported BVMOs are soluble proteins. This is in contrast to many other monooxygenase systems that often are found to be membrane-bound or membrane-associated. In 1997, Willetts concluded from careful inspection of... 

Zinc-containing alcohol dehydrogenases take up two electrons and a proton from alcohols in the form of a hydride. The hydride acceptor is usually NAD(P) (the oxidized form of nicotinamide adenine dinucleotide (NADH) or its phosphorylated derivative, NADPH). Several liver alcohol dehydrogenases have been structurally characterized, and Pig. 17.8 shows the environment around the catalytic Zn center and the bound NADH cofactor. 

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