Fungal riboflavin 5'-hydroxymethyl dehydrogenase catalyzes formation of both the aldehyde (riboflavinal) and the acid (riboflavinoic acid).
The purified enzyme from Schizophyllum commune that readily catalyzes the oxidation of the 5'-hydroxymethyl function of riboflavin (vitamin B2) with a redox dye and O2 to form the 5'-aldehyde can more slowly further oxidize the 5'-aldehyde to the 5'-acid. Hence, the formation of these so-called 'schizoflavins' can be accounted for by the action of one enzyme.
Flavin substrate specificity of the vitamin B2-aldehyde-forming enzyme from Schizophyllum commune.
Vitamin B2-aldehyde-forming enzyme from Schizophyllum commune catalyzes oxidation of the 5'-hydroxymethyl of riboflavin to the formyl group. We have monitored enzyme activity by spectrophotometrically measuring the reduction of 2,6-dichlorophenol-indolphenol as electron acceptor to assess 35 riboflavin analogs as potential substrates or competitive inhibitors with the purpose of delimiting structural requirements of the substrate binding site. Analogs with side chains of two- to six-carbon length modified by deletion of secondary hydroxyls or by changes in their epimeric configuration are not oxidized. The omega-hydroxyalkyl-flavins (n = 2-6) are competitive inhibitors (Ki = 7-16 microM) of riboflavin oxidation, as are some analogs with L-secondary hydroxyls in the side chain. Analogs with bulky substituents on the isoalloxazine ring are also not substrates. The enzyme does not significantly bind flavins with an 8 alpha-N-imidazole; diethylamino, methylethylamino, dimethylamino, ethylamino, or ethoxy groups at position 8; methyl at 6; and beta-hydroxyethylamino at position 2. Also the replacement of N with CH in 1-deazariboflavin disallows substrate reaction. Analogs with fluoro, chloro, methyl, amino, or methylamino at position 8; chloro at 7; methyl or carboxylmethyl at 3; thio at 2, and C replacing N at positions 3 or 5 are substrates with relative Vmax values ranging from 27 to 110% that of riboflavin. The Km values for the analogs oxidized are all found to be in the micromolar range (22-176 microM). Overall specificity of the enzyme for riboflavin is found to be rather narrow and sterically limited, which suggests that the vitamin is the natural substrate.
Affinity chromatographic purification and properties of flavokinase (ATP:riboflavin 5'-phosphotransferase) from rat liver.
Flavokinase (ATP:riboflavin 5'-phosphotransferase, EC 2.7.1.26) has been purified to apparent homogeneity from rat liver by affinity chromatography using flavinyl agarose beads (agarose-OCH2CONH(CH2)2NHCO(CH2)/N10-7,8-dimethylisoalloxazine). The specific activity of the pure enzyme is 9,900 units (nmol of FMN formed/h at 37 degrees C)/mg of protein, and reflects a one-step, 7000-fold purification. Flavokinase thus obtained, unlike previous preparations from mammalian sources, is free from contaminating phosphatase and FAD synthase. The purified enzyme rapidly loses activity upon storage but is stabilized by riboflavin and thiol-protecting reagents. The apparent molecular weight, estimated by gel filtration on Sephadex G-100 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is 28,000 +/- 1,000. Flavokinase phosphorylates and/or is inhibited by a large number of riboflavin analogs; however, the physiologically important 8 alpha-(amino acid)riboflavins are poorly accommodated. The strongly preferred phosphate donors are ATP and dATP. Both Zn2+ and Mg2+, as well as several other divalent cations, activate flavokinase, but Zn2+ yields greatest activity (1.8 times that with Mg2+). The pH optimum for activity with either Zn2+ or Mg2+ is approximately 9.3; at pH 7.0, the activity is 40% of that at the pH optimum.