Main Physicochemical Features of Monofunctional Flavokinase from Bacillus subtilis

I. M. Solovieva^*, K. V. Tarasov, and D. A. Perumov

Department of Molecular and Radiation Biophysics, Konstantinov St. Petersburg Institute of Nuclear Physics, Russian Academy of Sciences, Gatchina, Leningrad Region, 188350 Russia; fax: (812) 247-9191; E-mail: solovieva@em.uni-frankfurt.de

^* To whom correspondence should be addressed.

Received December 13, 2001; Revision received April 8, 2002

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The main properties of a monofunctional riboflavin kinase from B. subtilis have been studied for the first time; the enzyme is responsible for a key reaction in flavin biosynthesis--the ATP-dependent phosphorylation of riboflavin with production of flavin mononucleotide. The active form of the enzyme is a monomer with molecular weight of about 26 kD with a strict specificity for reduced riboflavin. To display its maximum activity, the enzyme needs ATP and Mg²⁺. During the phosphorylation of riboflavin, Mg²⁺ could be partially replaced by ions of other bivalent metals, the efficiencies of which decreased in the series Mg²⁺ > Mn²⁺ > Zn²⁺, whereas Co²⁺ and Ca²⁺ had inhibiting effects. The flavokinase activity was maximal at pH 8.5 and 52°C. ATP could be partially replaced by other triphosphates, their donor activity decreasing in the series: ATP > dATP > CTP > UTP. The Michaelis constants for riboflavin and ATP were 0.15 and 112 µM, respectively. As compared to riboflavin, a tenfold excess of its analog 7,8-dimethyl-10-(O-methylacetoxime)-isoalloxazine decreased the enzyme activity by 30%. Other analogs of riboflavin failed to markedly affect the enzyme activity.

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KEY WORDS: Bacillus subtilis, flavinogenesis, monofunctional flavokinase, physicochemical properties

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