Pyridine nucleotide dependent enzymes

Adams M (1987) Oxido-reductases - pyridine nucleotide-dependent enzymes. In Page MI, Wiliams A (eds) Enzyme mechanisms. The Royal Society of Chemistry, London, p 477... 

Pyridine nucleotide-dependent flavoenzyme catalyzed reactions are known for the external monooxygenase and the disulfide oxidoreductases However, no evidence for the direct participation of the flavin semiquinone as an intermediate in catalysis has been found in these systems. In contrast, flavin semiquinones are necessary intermediates in those pyridine nucleotide-dependent enzymes in which electron transfer from the flavin involves an obligate 1-electron acceptor such as a heme or an iron-sulfur center. Examples of such enzymes include NADPH-cytochrome P4S0 reductase, NADH-cytochrome bs reductase, ferredoxin — NADP reductase, adrenodoxin reductase as well as more complex enzymes such as the mitochondrial NADH dehydrogenase and xanthine dehydrogenase. 

MDase (L-malate NAD+ oxidoreductase EC 1.1.1.37), used in homogeneous EIA, is prepared from pig heart (mitochondria). The oxidation of L-malate is generally catalyzed by two distinct pyridine nucleotide-dependent enzymes those of the malate-oxaloacetate class, which use NAD+, and those of the malate-pyruvate class (commonly known as malic enzymes), which use NADP+ (Banaszak and Bradshaw, 1975). 

About 300 pyridine nucleotide dependent enzymes are currently known. Many of them are in widespread use for analytical purposes. Therefore the determination of the coenzyme NAD(P)H is of great importance. In contrast to the enzyme-catalyzed oxidation of NAD(P)H, its anodic oxidation proceeds in two separate one-electron steps with radical intermediates (Elving et al., 1982). It requires an overvoltage of about 1 V. Furthermore, electrode fouling by the reaction products makes the electrochemical process poorly reproducible. Owing to the high electrode potential, other oxidizable substances interfere signifi-... 

The oxidation of L-malate in most living organisms is catalyzed by two distinct types of pyridine nucleotide-dependent enzymes. In one case the principal product is oxaloacetate, while in the other it is pyruvate and CO2. The enzymes of the malate-oxaloacetate class, which utilize NAD+, have been referred to as simple dehydrogenases, while enzymes of the malate-pyruvate type, which, in contrast, use NADP+, have been designated decarboxylating dehydrogenases and are commonly known as malic enzymes (1). 

If El is a pyridine nucleotide independent dehydrogenase reducing medox to med d it can be reoxidized by an anode of an electrochemical cell or by an enzyme E3, which delivers the electrons to an acceptor such as dimethyl-sulphoxide or others. Pyridine nucleotide dependent enzymes El produce NADH or NADPH by the dehydrogenation of their substrates. The reduced pyridine nucleotides are reoxidized by the AMAPOR E2, which in turn reduces an artificial mediator medox to medred- Med d delivers electrons to E3 or an anode. For the reoxidation of E3 see text. 

In contrast the enzyme purified from lupin root nodule cytosol is reported to consist of a single polypeptide chain of molecular weight 235,000 (Boland and Benny, 1977). No information is available at present regarding the possible subunit structure of other eukaryote glutamate synthases although the ferredoxin-dependent enzyme from V. faba has a molecular weight of 150,000, as determined by gel filtration (Wallsgrove et al., 1977) and the pyridine nucleotide-dependent enzyme of Saccharomyces cerevisiae has a sedimentation coefficient of 14.6 S (Roon et al., 1974). 

Lynen had studied chemistry in Munich under Wieland his skill as a chemist led to the successful synthesis of a number of fatty acyl CoA derivatives which proved to be substrates in the catabolic pathway. Many of these C=0 or C=C compounds had characteristic UV absorption spectra so that enzyme reactions utilizing them could be followed spectrophotometrically. This technique was also used to identify and monitor the flavoprotein and pyridine nucleotide-dependent steps. Independent evidence for the pathway was provided by Barker, Stadtman and their colleagues using Clostridium kluyveri. Once the outline of the degradation had been proposed the individual steps of the reactions were analyzed very rapidly by Lynen, Green, and Ochoa s groups using in the main acetone-dried powders from mitochondria, which, when extracted with dilute salt solutions, contained all the enzymes of the fatty acid oxidation system. 

The stereospecificity of hydrogen transfer for estradiol-17 and estradiol-17(3 dehydrogenases has been examined by George et a/.84>. These enzymes are both present in chicken liver, and have substrates which differ only in the chirality of their substituents at C—17. Both of these enzymes were shown to use the 4-pro-S or 4B proton of the NADPH. Since the steroid is a bulky substrate, the authors argue that the steric fit between pyridine nucleotide and steroid cannot be as important as the role played by the enzyme in directing the fit. This paper contains an interesting summary of other recent work on the stereospecificity of pyridine nucleotide dependent-steroid dehydrogenases. 

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. 

The choice of the indicator electrode is largely determined by the species involved in the sensing reaction. Oxygen and H2O2, which are the cosubstrate and product of oxidases, as well as NAD(P)H—the cosubstrates of about 300 pyridine nucleotide-dependent dehydrogenases—can be determined amperometrically. Based on this principle, many enzyme sensors of the first generation have been developed and commercialized (see section 17.4 and table 17.2). 

A promising new area of enzymatic reactions is that of anaerobes. These organisms use redox systems that are not pyridine nucleotide dependent, but can use mcthylvirologen or benzylviologen (commercially available) as mediators. Electron donors can be hydrogen gas, formate, or carbon monoxide rather than glucose." The first new enzyme from this source is a 2-enoatc reductase, effected with stereospecific /rans-addition of hydrogen. An... 

For preparative work the so far applied 2-hydroxy carboxylate oxidoreductases are pyridine nucleotide dependent (l,2a,3). The substrate specificity of the individual representatives of the various oxidoreductases is rather narrow. For thermodynamical reasons the quantitative dehydrogenation of 2-hydroxy carboxylates by pyridine nucleotide dependent redox enzymes is not so easy. But such a reaction is interesting, too. For details see Section 3.2. 

It was of interest to purify and characterize the (R)-2-hydroxy carboxylate oxidore-ductase fi om Proteus vulgaris since it turned out that it does not react with NAD(H) or NADP(H) (46). Enzymes catalysing reversible reductions of carbonyl groups have been characterized fi om archaea, eubacteria and eukaryotes. The enzymes fi om eubacterial or eukaryotic sources depend on pyridine nucleotides the enzymes fi om archaea also use pyridine nucleotides or deazaflavin F42o- This coenzyme has a structure related to that of NAD(P)". Since viologens are suitable artificial electron mediators for this previously unknown enzyme we named it (/ )-2- droxy carboxylate viologen pxidoreductase (HVOR). In the purified form the membrane bound enzyme shows a specific activity of 1800 U mg protein for the reduction of phenylpyruvate (46). (One U (unit) reduces 1 pmol 2-0X0 carboxylate per minute). 

An Ordered Bi Bi system is fairly common in many enzyme reactions found in the nature, particularly among pyridine nucleotide-dependent dehydrogenases (Walsh, 1998). In this mechanism, there are two substrates A and B, and two products of reaction, P and Q, which add to the enzyme in an ordered fashion. 

Mammalian thioredoxin reductases are a family of selenium-containing pyridine nucleotide-disulfide oxidoreductases. These enzymes catalyze NADPH-dependent reduction of the redox protein thioredoxin (Trx), which contains a redox-active disulfide and dithiol group and by itself may function as an efficient cytosolic antioxidant [77]. One of the functions of Trx/ thioredoxin reductase system is the NADPH-catalyzed reduction of protein disulfide [78] ... 

The work by Scott and Lee 165) on the isolation of a crude enzyme system from a callus tissue culture of C. roseus was followed by studies of Zenk et al. on an enzyme preparation from a cell suspension system which produced indole alkaloids 166). The cell-free preparation was incubated with tryptamine and secologanin (34) in the presence of NADPH to afford ajmalicine (39), 19-epiajmalicine (92), and tetrahydroalstonine (55) in the ratio 1 2 0.5. No geissoschizine (35) was detected. In the absence of NADPH, an intermediate accumulated which could be reduced with a crude homogenate of C. roseus cells in the presence of NADPH to ajmalicine (39). Thus, the reaction for the formation of ajmalicine is critically dependent on the availability of a reduced pyridine nucleotide. 

This enzyme [EC 1.6.1.1] (also known as NAD(P)+ trans-hydrogenase (B-specific), pyridine nucleotide transhy-drogenase, and nicotinamide nucleotide transhydro-genase) catalyzes the reversible reaction of NADPH with NAD+ to produce NADP+ and NADH. This FAD-dependent enzyme is B-specific with respect to both pyridine coenzymes. In addition, deamino coenzymes will also serve as substrates. 

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