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J. Biol. Chem., Vol. 277, Issue 44, 41410-41416, November 1, 2002

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Biosynthesis of Riboflavin in Archaea Studies on the Mechanism of 3,4-Dihydroxy-2-butanone-4-phosphate Synthase of Methanococcus jannaschii^*

Markus Fischer^§, Werner Römisch, Susanne Schiffmann, Mark Kelly^¶, Hartmut Oschkinat^¶, Stefan Steinbacher, Robert Huber, Wolfgang Eisenreich, Gerald Richter, and Adelbert Bacher

From the  Institut für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstrasse 4, D-85747 Garching, Germany,  Department of Protein Crystallography, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18a, D-82512 Martinsried, Germany, and ^¶ Forschungsinstitut für Molekulare Pharmakologie, Robert-Rössle-Strasse 10, D-13125 Berlin, Germany

The hypothetical protein predicted by the open reading frame MJ0055 of Methanococcus jannaschii was expressed in a recombinant Escherichia coli strain under the control of a synthetic gene optimized for translation in an eubacterial host. The recombinant protein catalyzes the formation of the riboflavin precursor 3,4-dihydroxy-2-butanone 4-phosphate from ribulose 5-phosphate at a rate of 174 nmol mg¹ min¹ at 37 °C. The homodimeric 51.6-kDa protein requires divalent metal ions, preferentially magnesium, for activity. The reaction involves an intramolecular skeletal rearrangement as shown by ¹³C NMR spectroscopy using [U-¹³C₅]ribulose 5-phosphate as substrate. A cluster of charged amino acid residues comprising arginine 25, glutamates 26 and 28, and aspartates 21 and 30 is essential for catalytic activity. Histidine 164 and glutamate 185 were also shown to be essential for catalytic activity.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AF490541-AF490573 and AF516684.

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