Mononucleotide binding sites

Gervais, M., Labeyrie, F., Risler, Y. and Vergnes, O, 1980, A flavin-mononucleotide-binding site in Hcmsenula anomala nicked flavocytochrome hi, requiring the association of two domains. European Journal of Biochemistry 111, 17-31. 

Wang X, Mi G, Wang C, Zhang Y, Li J, Guo Y, Pu X, Li M (2012) Prediction of flavin mononucleotide binding sites using modified PSSM profile and ensemble support vector machine. Comput Biol Med 42( 11) 1053-1059... 

The C-terminal portion of the NOS protein closely resembles to cytochrome P-450 reductase, possesses many of the same cofactor binding sites, and basically performs the same functions. Consequently, this portion is often referred to as the reductase domain. At the extreme C-terminus is an NADPH binding region, which is conserved in all NOS and aligns perfectly with that of cytochrome P-450 reductase. The NADPH binding site is followed, in turn, by flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) consensus sequences. 

In 1989, BH4 was found to be a cofactor for nitric oxide synthase (NOS) [ 126, 127]. BH4 is also involved in dimerization of NOS, as NOS is catalytically active in a homodimer structure. Three isoforms of NOS exist neuronal NOS (NOS 1), inducible NOS (NOS 2) and endothelial NOS (NOS 3). BH4 is essential for all NOS isoforms. The NOS isoforms share approximately 50-60% sequence homology. Each NOS polypeptide is comprised of oxygenase and reductase domains. An N-terminal oxygenase domain contains iron protoporphyrin IX (heme), BH4 and an arginine binding site, and a C-terminal reductase domain contains flavin mononucleotide (FMN), and a reduced nicotin-amide adenine dinucleotide phosphate (NADPH) binding site. 

Some proteins contain other native fluorophores in addition to fluorescent amino acids. These include cofactors such as nicotinamide adenine dinucleotide (fluorescent in its reduced, NADH state) and flavin adenine dinucleotide (FAD). NADH is weakly fluorescent in water, but its fluorescence yield increases markedly on binding to a protein-binding site with an emission peak around 470 nm (3). FAD and flavin mononucleotide (FMN) are also fluorescent with an emission maximum around 520 nm, but fluorescence is quenched on binding to many flavoproteins (4). 

Fd is present in higher plants, algae, photosynthetic bacteria, and also in animals. It is an acidic protein with a pi of 4 and contains an [2Fe 2S]-type cluster with the rather low redox potential of about -410 mV at pH 7. The [2Fe 2S] cluster in Fd provides the site for one-electron redox change. It may be noted, however, that when algae are cultured in a medium lacking iron, a flavin-mononucleotide-binding redox protein called flavodoxin is produced as a replacement. 

Fig. 21. Stereogram ofthe nicotinamide binding site in the FNR(Y308->S)NADP complex. For clarity, only the isoalloxanzine and the ribityl portion of FAD and the nicotinamide mononucleotide portion of NADP are shown. Figure source Deng. Aliverti. Zanetti, Arakaki, Ottado, Orellano, Calcaterra, Ceccarelli, Carrillo and Karplus (1999) A productive NADP binding mode of ferredoxin-NADP reductase revealed by protein engineering and crystallographic studies. Nature Structural Biology 6 848...

The structure of the FMN-linked electron carrier, flavodoxin, has been determined from Clostridium MP by Ludwig and co-workers 59,60) and the corresponding protein from Desuljovibrio vulgaris by Jensen and coworkers (61). Rao and Rossmann 4) showed that, with the coordinates available to them, the standard deviation, o-, between these two structures this structure can be compared with the AMP mononucleotide binding unit of LDH and found that o- = 2.4 A for 39 equivalent atoms. When was 2.1 A for 102 equivalent Cn atoms. They also showed that part of these two quite different proteins were so aligned, it became clear that FMN binds to flavodoxin at a site similar to where AMP binds to LDH. Not only is it unlikely that this is an accidental structural relationship, but furthermore Baltscheffsky 62) has supported a common evolutionary origin for flavin and NAD+-linked enzymes on the basis of a comparison of their redox potentials. 

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... 

FIGURE I Schematic alignment of the deduced amino acid sequences of nitric oxide synthases (NOSs) and the cytochrome P-450 reductase. Depicted are consensus binding sites for heme, L-arginine, calmodulin (CaM), flavin mononucleotide (FMN), flavin-adenine dinucleotide (FAD), and NADPH. An NH2-terminal myristoylation site (myr) is present only in the endothelial constitutive NOS (ecNOS). n. Neuronal i, inducible. 

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