NADPH-dependent flavin

KMO is a NADPH-dependant flavin monooxygenase which is localized to the outer membrane of mitochondria [66]. KMO is expressed at high levels in the liver, endothelial cells, and monocytic cells and to a lower extent in the brain. Here, its expression is mainly found in cells of glial nature, specifically in microglial cells and in infiltrating macrophages, whereas little or no expression has been found in astrocytes or in neurons [12, 67]. However, the lack of good antibodies to detect... 

Phenol o-hydroxylase is an NADPH-dependent flavin mono-oxygenase which catalyses the oxidation of phenols to o-diphenols. Wainwright described a soil enzyme assay based on the colorimetric measurement of substrate (phenol) disappearance. Soils were incubated for Ih with phenol (O.OIM) in a citrate -phosphate buffer pH 4.0 (optimum) at 37°C (temperature optimum 40-45°C). Under these conditions soil activities were linear with time and the rates of reaction were proportional to the amounts of soil. Soil activities were stimulated by Cu (purified phenol o-hydroxylase has a Cu requirement). 

NADPH oxidation and NO synthesis by the enzyme, it supports a role for reduction of the heme iron in catalysis, and may explain why NOS functions only as an NADPH-dependent reductase in the absence of bound calmodulin (Klatt et ai, 1993). The mechanism of calmodulin gating is envisioned to involve a conformational change between the reductase and oxygenase domains of NOS, such that an electron transfer between the terminal flavin and heme iron becomes possible. Calmodulin may also have a distinct role within the NOS reductase domain, in that its binding dramatically increases reductase activity of the enzyme toward cytochrome c (Klatt et al., 1993 Heinzel et al., 1992). However, it is clear that several other NOS functions occur independent of calmodulin, including the binding of L-arginine and NADPH, and transfer of NADPH-derived electrons into the flavins (Abu-Soud and Stuehr, 1993). 

Fig. 7.4 Reactions associated with the thioredoxin system. Thioredoxin is a redox-regulating protein with a redox-active disulfide/dithiol within the conserved active site sequence -Cys-Gly-pro-Cys-. Thioredoxin reductase, a 55 kDa flavoprotein that catalyzes the NADPH-dependent reduction of thioredoxin (1) and thioredoxin oxidase (2), a flavin-dependent sulfhydry 1 oxidase that catalyzes the oxidative protein folding with the generation of disulfides...

In this process, an equivalent of glutathione disulfide is made and recycling of GSH through NADPH-dependent glutathione reductase [217] must be achieved for the protection to be maintained. Glutathione reductase is a flavin-containing enzyme which catalyzes the reduction of GSSG by NADPH as follows ... 

Treatment of E. coli sulfite reductase with p-mercuriphenyl sulfonate results in the specific release of FMN from the enzyme (390). FMN-depleted sulfite reductase can be prepared also by photodestruction of FMN. The enzyme-FMN dissociation constant is 10 nAf at 25°, and light irradiation can deplete the enzyme of FMN by destroying the released flavin. These treatments do not lead to removal or destruction of other components of the enzyme. The FMN-depleted enzyme is no longer capable of NADPH-dependent reduction of sulfite, nitrite, hydroxylamine. 

Nitrobenzene reductase activity has been detected in the fat body, gut, and Malpighian tubules of the Madagascar cockroach, G. portentosa (Rose and Young, 1973). Anaerobic conditions are essential for activity. The enzymes in the microsomes are strongly NADH dependent, whereas those in the soluble fraction are strongly NADPH dependent. Activity is enhanced by the addition of flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) or riboflavin. It appears that the true substrate for the nitroreductase is FMN and that the reduction of the nitro compounds occurs nonenzymatically (Figure 8.15). Similar results are obtained using azofuchsin as substrate. 

The second initiation way for the lipoperoxidation in the organism can be defined as semi-enzymatic or quasi-enzymatic. During this mechanism the O " radicals are generated by enzymes including NAD(P)H-dependent oxidases of mitochondrial and microsomal electron transport chaines, NADPH-dependent oxidase of phagocytes, xanthine oxidase and other flavine oxidases. After the HO formation the oxidation process develops in non-enzymatic way. 

Both the biosynthetic and degradative fatty acid cycles contain two oxidoreductases each. In the biosynthetic pathway, the /3-ketoacyl-ACP formed by the KAS enzymes is reduced by an NADPH-dependent reductase, encoded by the fabG gene in E. coli. Following a dehydration step, the resulting enoyl-ACP is reduced by an enoyl-ACP reductase, encoded by xS fahlin E. coli. FabI is an NADH-dependent reductase, and both FabI and FabG are members of the SDR superfamily. Not all bacteria utilize FabI as their enoyl-ACP reductase, and currently, three other enzymes that include FabV, FabL, and FabK are known. Both FabV and FabL are also members of the SDR family however, the flavin-dependent enoyl-ACP reductase FabK is not. 

The NOSs are best characterized as cytochrome P-450-like hemeprot-eins (Bredt et al., 1991 Stuehr and Ikeda, 1992 White and Marietta, 1992). They can be broadly divided into a reductase domain at the COOH terminus and an oxidative domain at the NH2 terminus (Fig. 1). The primary amino acid sequences of NOS isoforms share common consensus sequence binding sites for calmodulin, NADPH, flavin-adenine dinucleotide (FAD), and flavin mononucleotide (FMN) (Bredt et al., 1991 Marsden et al., 1992 Sessa et al., 1992 Xie et al., 1992 Lyons et al., 1992 Lowenstein et al., 1992). Each enzyme functions as a dimeric protein in catalyzing the NADPH-dependent five-electron oxidation of L-arginine to generate NO. L-Citrulline is a by-product (Back et al., 1993 Abu and Stuehr, 1993). Electrons are supplied by NADPH, transferred along the flavins and calmodulin, and presented to the catalytic heme center (Stuehr and Ikeda, 1992 White and Marietta, 1992). The NOS apoenzyme requires tetrahydrobiopterin, prosthetic heme (ferroprotoporphyrin IX), calmodulin, FMN, and FAD as cofactors for monomer assembly and/or catalytic activity (Abu and Stuehr, 1993 Mayer and Werner, 1994 Kwon etal., 1989 Stuehr and Ikeda, 1992 Stuehr and Griffith, 1992 White and Marietta, 1992 McMillan etal., 1992 Klatt... 

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