flavin-adenine-dinucleotide and pyridine

5-Hydroxypicolinic acid (5HPA), a natural pyridine derivative, is microbially degraded in the environment. However, the physiological, biochemical, and genetic foundations of 5HPA metabolism remain unknown. In this study, an operon (

Topics: Alcaligenes faecalis; Amidohydrolases; Bacterial Proteins; Biodegradation, Environmental; Flavin-Adenine Dinucleotide; Hydroxylation; Kinetics; Mixed Function Oxygenases; Operon; Phylogeny; Pyridines

Flavocytochrome P450 BM3 is a member of the diflavin reductase enzyme family. Members include cytochrome P450 reductase, nitric-oxide synthase, methionine synthase reductase, and novel oxidoreductase 1. These enzymes show a strong preference for NADPH over NADH as reducing coenzyme. An aromatic residue stacks over the FAD isoalloxazine ring in each enzyme, and in some cases it is important in controlling coenzyme specificity. In P450 BM3, the aromatic residue inferred from sequence alignments to stack over the FAD is Trp-1046. Mutation to Ala-1046 and His-1046 effected a remarkable coenzyme specificity switch. P450 BM3 W1046A/W106H FAD and reductase domains are efficient NADH-dependent ferricyanide reductases with selectivity coefficients (k(cat)/K(m)(NADPH)/k(cat)/K(m)(NADH)) of 1.5, 67, and 8571 for the W1046A, W1046H, and wild-type reductase domains, respectively. Stopped-flow photodiode array absorption studies indicated a charge-transfer intermediate accumulated in the W1046A FAD domain (and to a lesser extent in the W1046H FAD domain) and was attributed to formation of a reduced FADH(2)-NAD(P)(+) charge-transfer species, suggesting a relatively slow rate of release of NAD(P)(+) from reduced enzymes. Unlike wild-type enzymes, there was no formation of the blue semiquinone species observed during reductive titration of the W0146A/W146H FAD and reductase domains with dithionite or NAD(P)H. This was a consequence of elevation of the semiquinone/hydroquinone couple of the FAD with respect to the oxidized/semiquinone couple, and a concomitant approximately 100-mV elevation in the 2-electron redox couple for the enzyme-bound FAD (-320, -220, and -224 mV in the wild-type, W1046A, and W1046H FAD domains, respectively).

Topics: Alanine; Amino Acid Substitution; Animals; Bacillus megaterium; Bacterial Proteins; Cytochrome P-450 Enzyme System; Flavin-Adenine Dinucleotide; Histidine; Kinetics; Mixed Function Oxygenases; Mutation; NADP; NADPH-Ferrihemoprotein Reductase; Potentiometry; Protein Structure, Tertiary; Pyridines; Rats; Spectrophotometry; Substrate Specificity; Thermodynamics; Tryptophan

The interaction between glucose oxidase (GOx) and a typical metal complex, which is chemically stable in both oxidized and reduced forms, has been investigated by a voltammetric method. The evaluation of an electron-transfer mediator useful for glucose oxidation is discussed from thermodynamic and kinetic points of view, i.e. the redox potentials of various metal complexes and the second-order rate constants for the electron transfer between GOx in reduced form and the metal complexes in oxidized form. No mediation of glucose oxidation by [Co(bpy)(3)](2+) (bpy=2,2'-bipyridine) or [Cu(bpy)(2)](2+) occurred, in spite of their appropriate redox potentials. This was attributed mainly to the lower electron-self-exchange rates of the mediator and the reaction with GOx. All three types of osmium(II) complexes, [Os(PP) (n)](2+) ( n=2 or 3; PP=polypyridine), [OsL(2)(PP)(2)](2+) (L=imidazole and its derivatives), and [OsClL(bpy)(2)](+), acted as excellent electron-transfer mediators for the glucose oxidation. Mixed ligand complexes, [OsL(2)(PP)(2)](2+) and [OsClL(bpy)(2)](+), have been concluded to be more efficient electron-transfer mediators. The electron-transfer rates between the mediator and GOx have been found to be accelerated by intermolecular electrostatic interactions or hydrogen bonds.

Topics: Electrochemistry; Flavin-Adenine Dinucleotide; Glucose; Glucose Oxidase; Kinetics; Ligands; Metals; Organometallic Compounds; Osmium Compounds; Osmolar Concentration; Oxidation-Reduction; Pyridines; Sodium Chloride

Assimilatory NADH:nitrate reductase (EC 1.6.6.1), a complex Mo-pterin-, cytochrome b557-, and FAD-containing protein, catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by high plants. With a recombinant, histidine-tagged form of the spinach nitrate reductase flavin domain, site-directed mutagenesis has been utilized to examine the role of lysine 741 in binding the reducing substrate, NADH. Seven individual mutants, corresponding to K741R, K741H, K741A, K741E, K741M, K741Q, and K741P, have been engineered and six of the resulting proteins purified to homogeneity. With the exception of K741P, all the mutants were obtained as functional flavoproteins which retained FAD as the sole prosthetic group and exhibited spectroscopic properties comparable to those of the wild-type domain, indicating that the amino acid substitutions had no effect on FAD binding. In contrast, all the mutants were found to have altered NADH:ferricyanide reductase (NADH:FR) activity with mutations affecting both kcat and K(NADH)m, which decreased and increased, respectively. At pH 7.0, kcat decreased in the order WT > K741R > K741A > K741H > K741E > K741M > K741Q while K(NADH)m increased in the same order. The most efficient mutant, K741R, retained 80% of the wild-type NADH:FR activity, while in contrast the most inefficient mutant, K741Q, retained only 18% of the wild-type NADH:FR activity together with a 118-fold increased K(NADH)m. pH studies of K741H revealed that both kcat and K(NADH)m were pH-dependent, with enhanced activity observed at acidic pH. These results indicated that retention of a positively charged side chain at position 741 in the spinach nitrate reductase primary sequence is important for the efficient binding and subsequent oxidation of NADH and that the positively charged side chain enhances nucleotide binding via charge complementarity with the negatively charged pyrophosphate moiety.

Topics: Amino Acid Sequence; Amino Acid Substitution; Circular Dichroism; Flavin-Adenine Dinucleotide; Hydrogen-Ion Concentration; Kinetics; Lysine; Molecular Sequence Data; Mutation; NAD; NADH, NADPH Oxidoreductases; Nitrate Reductase; Nitrate Reductases; Protein Structure, Tertiary; Pyridines; Sequence Alignment; Spectrometry, Fluorescence; Spectrum Analysis; Spinacia oleracea; Static Electricity

Topics: Coenzymes; Flavin-Adenine Dinucleotide; Liver; Nucleosides; Nucleotides; Pyridines; Thiamine Pyrophosphate; Vitamin B 12