The "super mutant" of yeast FMN adenylyltransferase enhances the enzyme turnover rate by attenuating product inhibition.

FMN adenylyltransferase (FMNAT) is an essential enzyme catalyzing the last step of a two-step pathway converting riboflavin (vitamin B2) to FAD, the ubiquitous flavocoenzyme. A structure-based mutagenesis and steady-state kinetic analysis of yeast FMNAT unexpectedly revealed that mutant D181A had a much faster turnover rate than the wild-type enzyme. Product inhibition analysis showed that wild-type FMNAT is strongly inhibited by FAD, whereas the D181A mutant has an attenuated product inhibition. These results provide a structural basis for the product inhibition of the enzyme and suggest that product release may be the rate-limiting step of the reaction.

Azo reduction of methyl red by neuronal nitric oxide synthase: the important role of FMN in catalysis.

Nitric oxide synthase (NOS) is composed of an oxygenase domain and a reductase domain. The reductase domain has NADPH, FAD, and FMN binding sites. Wild-type nNOS reduced the azo bond of methyl red with a turnover number of approximately 130 min(-1) in the presence of Ca(2+)/calmodulin (CaM) and NADPH under anaerobic conditions. Diphenyleneiodonium chloride (DPI), a flavin/NADPH binding inhibitor, completely inhibited azo reduction. The omission of Ca(2+)/CaM from the reaction system decreased the activity to 5%. The rate of the azo reduction with an FMN-deficient mutant was also 5% that of the wild type. NADPH oxidation rates for the wild-type and mutant enzymes were well coupled with azo reduction. Thus, we suggest that electrons delivered from the FMN of the nNOS enzyme reduce the azo bond of methyl red and that this reductase activity is controlled by Ca(2+)/CaM.

Development of a simple antioxidant screening assay using human skin fibroblasts.

The purpose of this study was to develop a simple antioxidant screening assay for quantifying the protective effects of antioxidant enzymes, inhibitors and scavengers against extracellularly generated oxygen species on human skin fibroblast cytotoxicity. Different in vitro oxidative stresses have been studied: xanthine oxidase-hypoxanthine, flavin mononucleotide-NADH, and hydrogen peroxide. Cytotoxicity and protection were evaluated by two procedures: evaluation of the living cells using a colorimetric method (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT), and ability of the viable cells to adherate and proliferate. Hypoxanthine-xanthine oxidase and H2O2 induced a dose dependent cytotoxicity only when we considered the delayed toxicity. The influence of the cell density was also investigated. The delayed toxicity was higher when cell density increased. One hundred percent protection against free radical cytotoxicity induced by the three systems were obtained with catalase (500 U/ml). When the oxidative stress used was H2O2 90-96% protection was obtained with deferoxamine an iron chelating agent that prevents iron catalysed radical reactions. Using the colorimetric method no significant protection was obtained when SOD was added before and during the stresses. Using the fibroblasts ability to proliferate SOD (10-150 micrograms/ml) reduced xanthine oxidase (20 U/l)-hypoxanthine (0.10-0.30 mM) or H2O2 (1-6 mM) cytotoxicity by 15-20%. SOD did not act as antioxidant when the applied stress was mediated by flavin. In this study we showed a paradoxical effect and the cytotoxicity of flavin-NADH system increased when we added SOD to the cell medium. This simple and reliable antioxidant screening assay required no costly or radioactive equipment.