NAD dependent oxidoreductases

The pharmaceutical and fine chemical industry might use pure hydrogenase or partially purified enzyme preparations in bioconversion applications such as regio and stereoselective hydrogenation of target compounds (van Berkel-Arts et al. 1986). Enzymes are able to catalyse such stereospecific syntheses with ease. However, the cofactors for the NAD-dependent oxidoreductases are expensive. The pyridine nucleotide-dependent hydrogenases such as those from Ralstonia eutropha and hyperthermophilic archaea (Rakhely et al. 1999) make it possible to exploit H2 as a low-cost reductant. The use of inverted micelles in hydrophobic solvents, in which H2 is soluble, has advantages in that the enzymes appear to be stabilized. 

Figure 15. Hypothetical scheme for the transformations experienced by veratrylglycerol-B-guaiacyl ether at the hands of a seemingly extracellular NAD-dependent oxidoreductase system from Poria subacida (90).

This NAD+-dependent oxidoreductase [EC 1.1.1.27] catalyzes the following reversible reaction ... 

Recently, it has been shown that there are NAD-dependent oxidoreductases that will not liberate NADH/NAD+ from the active site. They catalyse such redox reactions, albeit not with formate but with less favourable electon donors and with low rates [9]. When these enzymes can be properly engineered and produced, they will impose few constraints on the reactor design. This situation is analogous to what has been described in the previous section for synthetic reactions using hydrolases if a biocatalyst is found that can directly convert the substrates into the desired products, without formation of intermediates or occurrence of side-reactions, the reactor design becomes simple. 

More recently, the focus has been put on formal nucleophilic substitution of —OH or —NH2 groups. To perform this biocatalytic variant of the Mitsunobu reaction, an oxidation-nucleophilic addition-reduction sequence is necessary, for which linked NAD-dependent oxidoreductases are ideally suited. The early contributions from the Forschungszentrum Jiilich [79] have been recently rediscovered by Kroutil and coworkers [80]. By combining a mandelate racemase (MR) with a mandelate dehydrogenase and an L-amino acid dehydrogenase, the authors could completely transform racemic mandelic acid into enantiopure (S)-phenyl-glycine (Scheme 8.16). 

Glucose-grown cells of G. candidum SC 5469 have also catalyzed die stereoselective reduction of ethyl, isopropyl, and terdary butyl esters of 4-chloro-3-oxobutanoic acid and both methyl and ethyl esters of 3-bromo-3-oxobutanoic acid. A reaction yield of more than 85% and e.e. s of more than 94% were obtained. NAD -dependent oxidoreductase responsible for the stereoselective reduction of P-keto esters of 4-chloro- and 4-bromo-3-oxobutanoic acid was purified 100-fold. The molecular weight of purified enzyme is 950,000. The purified oxidoreductase was immobilized on Eupergit C and used to catalyze the reduction of 52 to 5-(—)-53. The cofactor NAD required for the reduction reaction was regenerated by glucose dehydrogenase. 

The aldehyde dehydrogenases are members of a superfamily of pyridine nucleotide [NAD(P)+]-dependant oxidoreductases that catalyze the oxidation of aldehydes to... 

Devaux-Basseguy, R., Bergel, A. and Comtat, M., Potential applications of NAD(P)-dependent oxidoreductases in s3fnthesis a survey. Enzyme Microb. TechnoL, 1997, 20, 248. 

In this pathway the electrons for the drug reduction are generated by the oxidative decarboxylation of malate catalyzed by the NAD-dependent malic enzyme (malate dehydrogenase (decarboxylating)). The NADH produced by this reaction is reoxidized by an enzyme with NADH ferredoxin oxidoreductase activity that has been recently identified as a homologue of the NADH dehydrogenase (NDH) module of the mitochondrial respiratory complex I (Hrdy et al. 2004 and see Hrdy et al., this volume). The... 

NAD(P)H is required in the bioreductions of many xenobiotic compounds. However, NADH or NADPH are not necessarily the entities that directly react with the organic substrate. Rather, the prosthetic groups of other enzymes are themselves reduced by NAD(P)H, and the resulting reduced enzyme components are the actual reactants involved in bond making and breaking of the xenobiotic substance. A prominent set of examples involves the flavin-dependent oxidoreductases. The key reactive portion in these flavoproteins is the three-ring flavin (FAD) which is reduced by NAD(P)H to FADH2 ... 

In Leuconostoc oenos ML 34, we have shown oxaloacetic acid decarboxylation manometrically (6, 7, 8). We were also able to demonstate fluorometrically the enzymatic production of reduced NAD with malic acid as a substrate, but, of course, were unable to do so with oxaloacetic acid since no NADH could be formed from this substrate. It is likely that this oxaloacetic acid decarboxylation activity, as in Lactobacillus plantarum, is distinct from the activity causing the malic-lactic transition. It is also possible that oxaloacetic acid decarboxylation is caused by a malic enzyme. However, there is no verified NAD dependent malic oxidoreductase (decarboxylating) enzyme which does so (12). For example, Macrae (31) isolated a malic enzyme from cauliflower bud mitochondria which showed no activity with oxaloacetic acid. Similarly, Saz (32) isolated a malic enzyme from Ascaris lumbricoides which is also inactive toward oxaloacetic acid. True, the Enzyme Commission (12) lists an enzyme described as L-malate NAD oxidoreductase (decarboxylating) (E.C. 1.1.1.38) which is said to be capable of decarboxylating oxaloacetic acid, but its description dates back to the studies of Ochoa and his group, and we now feel this listing may be improper. 

W. Hummel, Large-scale applications of NAD(P)-dependent oxidoreductases recent developments, Trends Biotechnol. 1999, 37, 487-492. 

The second step in the synthesis of bile acids, according to Hylemon et al. (1991), is the conversion of 7a-hydroxycholesterol to 7a-hydroxy-4-cholesten-3-one by NAD+-dependent 3/3-hydroxy-A5-C27-steroid oxidoreductase. This enzyme is located in the endoplasmic reticulum of liver, and its catalysis of the 3/3-hydroxy group also results in isomerization of the double bond from A5 to A4. 

Diverse soluble enzymes, called aldo-keto reductases. cany out bioreduction of aldehydes and ketones. They are found in the liver and other tissues (e.g.. kidney). As a general class, these soluble en7.ynie.s have similar physi-ochemical properties and broad substrate specificities and require NADW as a cofactor. Oxidoreductase enzymes that catty out both oxidation and reduction reactions also can reduce aldehydes and ketones. " For example. Ihe important liver alcohol dehydrogenase is an NAD -dependent oxido-icductase that oxidizes ethanol and other aliphatic alcohols to aldehydes and ketones. In the presence of NADH or... 

Figure 3-7. Sequence alignment of various enzymes in the flavopro-tein disulfide oxidoreductase family. The sequences of the NADP4-dependent enzymes are the glutathione reductase from E. coli (E-GR), human (H-GR), Pseudomonas aeruginosa (P-GR), mercuric reductase from Staphylococcus aureus (S-MR), P. aeruginosa Tn 501 (P-GR), and trypanothione reductase from Trypanosoma congolense (T-TR). The NAD+-dependent enzymes are dihydrolipoamide dehydrogenase from E. coli (E-DD), B. stearothermophilus (B-DD), yeast (Y-DD), and human (H-DD). Residue positions marked with an asterisk correspond to those that were targets of site-directed mutagenesis in the text.

HLADH is certainly one of the most prominent and widely used oxidoreductases. The NAD-dependent enzyme is a dimer consisting of two almost identical subunits,... 

Most NAD(P)-dependent oxidoreductases, dehydratases, and epimerases are members of either the aldo-keto reductase (AKR) superfamily or the SDR superfamily. Proteins in the AKR family share a common (a//3)g-barrel fold and are involved in the metabolism of many important compounds including aldehydes, sugars, and... 

Pyruvate dehydrogenase of propionibacteria differs from that of aerobic bacteria in that it does not depend on lipoate and has a similarity with pyruvate cytochrome b oxidoreductase. Castberg and Morris (1978) reported the isolation from cells of P. shermanii of pyruvate oxidase (reducing 2,6-dichlorophenolindophenol (DCIP)) and pyruvate dehydrogenase system (reduces NAD). The pyruvate oxidase could not use NAD as an electron acceptor, and the NAD-dependent enzyme did not transport electrons to DCIP. Unlike the pyruvate oxidase of E. coli, the enzyme from P, shermanii was not activated by phosphatydylcholine in the presence of SDS, and the presence of thiamine diphosphate and Mg " was not required for the activity of purified preparations. 

---