A novel formamidase is required for riboflavin biosynthesis in invasive bacteria

Biosynthesis of riboflavin, the precursor of the redox cofactors FMN and FAD, was thought to be well understood in bacteria, with all the pathway enzymes presumed to be known and essential. Our previous research has challenged this view by showing that, in the bacterium Sinorhizobium meliloti, deletion of the ribBA gene encoding the enzyme that catalyzes the initial steps on the riboflavin biosynthesis pathway only causes a reduction in flavin secretion rather than riboflavin auxotrophy. This finding led us to hypothesize that RibBA participates in the biosynthesis of flavins destined for secretion, while S. meliloti has another enzyme that performs this function for internal cellular metabolism. Here, we identify and biochemically characterize a novel formamidase (SMc02977) involved in the production of riboflavin for intracellular functions in S. meliloti. This catalyst, which we named Sm-BrbF, releases formate from the early riboflavin precursor 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (AFRPP) to yield 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (DARoPP). We show that homologs of this enzyme are present in many bacteria, are highly abundant in the Rhizobiales order, and that sequence homologs from Brucella abortus and Liberobacter solanacearum complement the riboflavin auxotrophy of the Sm1021ΔSMc02977 mutant. Furthermore, we show that the B. abortus enzyme (Bab2_0247, Ba-BrbF) is also an AFRPP formamidase, and that the bab2_0247 mutant is a riboflavin auxotroph exhibiting a lower level of intracellular infection than the wild-type strain. Finally, we show that Sm-BrbF and Ba-BrbF directly interact with other riboflavin biosynthesis pathway enzymes. Together, our results provide novel insight into the intricacies of riboflavin biosynthesis in bacteria.