Catalysis of an Essential Step in Vitamin B 2 Biosynthesis by a Consortium of Broad Spectrum Hydrolases

An enzyme catalysing the essential dephosphorylation of the riboflavin precursor, 5‐amino‐6‐ribitylamino‐2,4(1 H ,3 H )‐pyrimidinedione 5′‐phosphate ( 6 ), was purified about 800‐fold from a riboflavin‐producing Bacillus subtilis strain, and was assigned as the translation product of the ycs E gene by mass spectrometry. YcsE is a member of the large haloacid dehalogenase (HAD) superfamily. The recombinant protein was expressed in Escherichia coli . It catalyses the hydrolysis of 6 ( v max , 12 μmol mg −1  min −1 ; K M , 54 μ m ) and of FMN ( v max , 25 μmol mg −1  min −1 ; K M , 135 μ m ). A ycs E deletion mutant of B. subtilis was not riboflavin dependent. Two additional proteins (YwtE, YitU) that catalyse the hydrolysis of 6 at appreciable rates were identified by screening 13 putative HAD superfamily members from B. subtilis . The evolutionary processes that have resulted in the handling of an essential step in the biosynthesis of an essential cofactor by a consortium of promiscuous enzymes require further analysis.