Flavin mononucleotide oxidations using

Semisynthetic enzymatic oxidation of peptide alcohols employs equine liver alcohol dehydrogenase. Amino alcohols with nonpolar side chains and Z-Om[CH2OH] worked as effective substrates while polar amino alcohols such as H-Arg[CH2OH] and H-Lys[CH2OH] failed as substrates. To attain complete oxidation, semicarbazide was present in the reaction mixture to immediately trap the aldehyde, and flavin mononucleotide was used to oxidize the NADH to NAD+, which serves to oxidize the alcohol 41] Configurational stability was confirmed by NMR spectroscopy as in the case of Ac-Phe[CH2OH], which was prepared by sodium borohydride reduction of Ac-Phe-H 4 1... 

Pollock RJ, LB Hersh (1973) A-methylglutamate synthetase. The use of flavin mononucleotide in oxidative catalysis. J Biol Chem 248 6724-6733. 

A still more complicated reaction is the chemiluminescent oxidation of sodium hydrogen sulfide, cysteine, and gluthathione by oxygen in the presence of heavy metal catalysts, especially copper ions 60>. When copper is used in the form of the tetrammin complex Cu(NH3) +, the chemiluminescence is due to excited-singlet oxygen when the catalyst is copper flavin mononucleotide (Cu—FMN), additional emission occurs from excited flavin mononucleotide. From absorption spectroscopic measurements J. Stauff and F. Nimmerfall60> concluded that the first reaction step consists in the addition of oxygen to the copper complex ... 

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. 

Vitamin B2 (riboflavin) acts mainly as the coenzyme FAD (flavin adenine nucleotide) rmd FMN (flavin mononucleotide), which are used in many oxidation-reduction reactions in which hydrogen atoms are received or donated. Particularly noteworthy examples are their uses in... 

Reactions with HLADH typically occur at temperatures between 4°C and 25°C and in the pH range of 5 to 10. For catalysis of a reduction the optimum pH is 7 while for the reverse oxidation it is 8. Reaction times vary from a few hours in the most favourable substrates and 2-3 weeks for the slowest. The disadvantage of HLADH has been the high cost of coenzymes. Fortunately, several recycling methods are available that allow reduction of substrates at the research scale (up to 1 kg of substrate).27-30 Por example, the ethanol-coupled method has been used for reduction and flavin mononucleotide (FMN) recycling for oxidation. 

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 final step in this pathway is the oxidation of PNP to PLP and is carried out by PdxH. Tbe recombinant enzyme from E. coli has been studied in vitro and is a 51 kDa homodimer that utilizes flavin mononucleotide (FMN) as a cofactor. PdxH can use either PNP or pyridoxamine 5 -phosphate (PMP) as a substrate with a of 2 and 105 pM and cat of 0.8 and 1.7 s for PNP and PMP, respectively. The structures of the enzyme from E. coli as well as homologues from Mycobacterium tuberculosis and humans have been solved. The E. coli enzyme with PLP and FMN bound is shown in Figure 6. PdxH is involved in both the biosynthetic and the salvage pathways and is further discussed in a section describing the transport, salvage, and interconversion of the various forms of vitamin Bg. 

Aflavoprotein is an enzyme that contains either flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) as a coenzyme. FAD and FMN, like NAD and NADP, are coenzymes used in oxidation reactions. As its name indicates, FAD is a dinucleotide in which one of the heterocyclic components is flavin and the other is adenine. FMN contains flavin but not adenine—it is a mononucleotide. (Flavin is a bright yellow compound flavus is Latin for yellow. ) Notice that instead of ribose, the flavin nucleotide has a ribitol group (a reduced ribose). Flavin plus ribitol is called riboflavin. Riboflavin is also known as vitamin B2. A vitamin B2 deficiency causes inflammation of the skin. 

As for reduction processes, C02 free radicals were shown to react specifically with disulfide bonds (122). They were extensively used to study the redox properties of disulfide bonds, thiyl and disulfide free radicals in proteins. This is discussed in paragraph 5. However, they do react with thiol functions also (37). For proteins containing a prosthetic group, the reduction concerns also oxidized valencies of metals and flavins. Flavin adenine dinucleotide (FAD) or Flavin Mononucleotide (FMN). The proportion of reduced disulfide/reduced prosthetic group varies considerably with the protein. For instance, lipoamide dehydrogenase contains one disulfide bond close to a flavin (FAD). Free radicals can reduce only the flavin, although both are in the active site (123). In chicken egg white riboflavin binding protein, competitive formation of both disulfide and semireduced flavin is observed (124). 

FIGURE 15.4 The structures of riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). Even in organisms that rely on the nicotinamide coenzymes (NADH and NADPH) for many of their oxidation-reduction cycles, the flavin coenzymes fill essential roles. Flavins are stronger oxidizing agents than NAD and NADP. They can be reduced by both one-electron and two-electron pathways and can be reoxidized easily by molecular oxygen. Enzymes that use flavins to carry out their reactions—flavoenzymes—are involved in many kinds of oxidation-reduction reactions. 

Riboflavin (vitamin B2) also acts as a cofactor and is a precursor for the coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). These coenzymes are used in metabolism and catalyze numerous oxidation—reduction reactions. Among the good dietary sources for riboflavin, most animal-derived products, milk and dairy products, are pointed out. Foods are usually pretreated before analysis of riboflavin following similar procedures to those described for vitamin Bi. Similarly, fluorescence detection is mostly employed (370 nm ex., 520 ran em.) after RP separation. 

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