Flavin mononucleotide function

The flavin-based coenzymes flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) are associated with a wide variety of enzymes that catalyze reactions in critical biosynthetic and catabolic processes (Fig. 16). Unlike other coenzymes, the reactions catalyzed do not conserve specific mechanistic pathways. In each case the apoenzyme serves to steer the course of the reaction through specific interactions with substrate and coenzyme [55]. Nonetheless, there are common features of the interactions of the apoenzymes with the flavin which can be exploited in the design of functional peptides and proteins. 

It carries its physiological function in its active forms, flavin mononucleotide (FMN) and flavin adenine dinucleotide. These coenzymes are involved in various biochemical reactions. 

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. 

Flavin adenine dinucleotide (FAD) (fig. 10.8) and flavin mononucleotide (FMN) are the coenzymatically active forms of vitamin B2, riboflavin. Riboflavin is the NI0-ribityl isoalloxazine portion of FAD, which is enzymatically converted into its coenzymatic forms first by phosphorylation of the ribityl C-5 hydroxy group to FMN and then by ade-nylylation to FAD. FMN and FAD are functionally equivalent coenzymes, and the one that is involved with a given enzyme appears to be a matter of enzymatic binding specificity. 

A model of a flavin-based redox enzyme was prepared.[15] Redox enzymes are often flavoproteins containing flavin cofactors flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN). They mediate one- or two-electron redox processes at potentials which vary in a range of more than 500 mV. The redox properties of the flavin part must be therefore tuned by the apoenzyme to ensure the specific function of the enzyme. Influence by hydrogen bonding, aromatic stacking, dipole interactions and steric effects have been so far observed in biological systems, but coordination to metal site has never been found before. Nevertheless, the importance of such interactions for functions and structure of other biological molecules make this a conceivable scenario. 

Oxidation of NADH begins with complex I, also termed NADH dehydrogenase or NADH ubiquinone oxidoreductase. It contains 25 polypeptide chains, flavine mononucleotide (FMN), and several iron-sulfur centers. The function of this complex is to reduce a substance called ubiquinone (UQ or CoQ), whose structure is shown in Figure 17.5. UQ is not protein bound and can move about freely. In the process of reducing UQ, the NADH is oxidized to NAD+. It is now accepted that in complex I, NADH first reduces FMN, and the resulting FMNH2 then transfers its electrons through at least three iron-sulfur centers to UQ. As the electrons pass from NADH to UQ, two to four protons are extruded from the mitochondrial matrix across the inner membrane. 

Once the hexameric structure of the yeast FAS was established, the number of functional active sites still remained to be determined. Earlier studies had shown that the functional complex contains approximately six equivalents each of two prosthetic groups 4 -phosphopantetheine [60,63], necessary for the AGP functionality, and flavin mononucleotide [64], an essential component of the enoyl reductase activity. These studies provided an early indication that each of the six active sites in the complex has a full set of the chemical groups necessary for fatty acid synthesis. Nevertheless, conflicting reports appeared in the literature as to the competence of six active sites. Whereas some reports suggested the possibility of half-sites reactivity (only three of the six sites are catalytically competent) [65, 66], others proposed that all six active sites could synthesize fatty acids [62]. Subsequent active site titration experiments were performed which quantitated the amount of fatty acyl products formed in the absence of turnover [67]. Single-turnover conditions were achieved through the use of... 

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. 

In 1915, Harden and Norris observed that dried yeast, when mixed with lactic acid, reduced methylene blue and formed pyruvic acid 4). Thirteen years later Bernheim prepared an extract from acetone-dried baker s yeast, which had lactate dehydrogenase activity (5). Bach and co-workers demonstrated that the lactate dehydrogenase activity was associated with a 6-type cytochrome, which they named cytochrome 62 (6). In 1954, the enzyme was crystallized, enabling the preparation of pure material and the identification of flavin mononucleotide as a second prosthetic group (2). Since then, significant advances have been made in the analysis of the structure and function of the enzyme. Much of the earlier work on flavocytochrome 62 has already been summarized in previous review articles (7-10). In this article we shall describe recent developments in the study of this enzyme, ranging fi om kinetic, spectroscopic, and structural data to the impact of recombinant DNA technology. 

In higher mammals, riboflavin is absorbed readily from the intestines and distributed to all tis.sues. It is the precursor in the biosynthesis of the cocnzyme.s flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). The metabolic functions of this vitamin involve these Iwocoenzymes. which participate in numerous vital oxidation-reduction proces.ses. FMN (riboflavin 5 -phosphate) is produced from the vitamin and ATP by flavokinasc catalysis. This step con be inhibited by phcnothiazincs and the tricyclic antidepressants. FAD originates from an FMN and ATP reaction that involves reversible dinucicotide formation catalyzed by flavin nucleotide pyrophosphorylase. The.se coenzymes function in combination with several enzymes as coenzyme-en-zyme complexes, often characterized as, flavoproteins. 

Whereas redox reactions on metal centres usually only involve electron transfers, many oxidation/reduction reactions in intermediary metabolism, as in the case above, involve not only electron transfer, but hydrogen transfer as well — hence the frequently used denomination dehydrogenase . Note that most of these dehydrogenase reactions are reversible. Redox reactions in biosynthetic pathways usually use NADPH as their source of electrons. In addition to NAD and NADP+, which intervene in redox reactions involving oxygen functions, other cofactors like riboflavin (in the form of flavin mononucleotide, FMN, and flavin adenine dinucleotide, FAD) (Figure 5.3) participate in the conversion of [—CH2—CH2— to —CH=CH—], as well as in electron transfer chains. In addition, a number of other redox factors are found, e.g., lipoate in a-ketoacid dehydrogenases, and ubiquinone and its derivatives, in electron transfer chains. 

The answer is b. (Murray, pp 627-661. Scriver, pp 3897-3964. Sack, pp 121-138. Wilson, pp 287-320.) Nicotinamide adenine dinucleotide (NAD+) is the functional coenzyme derivative of niacin. It is the major electron acceptor in the oxidation of molecules, generating NADH, which is the major electron donor for reduction reactions. Thiamine (also known as vitamin Bi) occurs functionally as thiamine pyrophosphate and is a coenzyme for enzymes such as pyruvate dehydrogenase. Riboflavin (vitamin B2) functions in the coenzyme forms of flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD). When concentrated, both have a yellow color due to the riboflavin they contain. Both function as prosthetic groups of oxidation-reduction enzymes or flavoproteins. Flavoproteins are active in selected oxidation reactions and in electron transport, but they do not have the ubiquitous role of NAD+. 

Redox-active cofactors are important species in biological systems, playing vital roles in redox and electron-transfer processes. Among the structurally and functionally diverse redox enzymes, flavoproteins containing the flavin cofactors flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) are involved in many different biochemical processes serving as a highly versatile redox... 

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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