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Title: A novel formamidase is required for riboflavin biosynthesis in invasive bacteria

item Yurgel, Svetlana
item JOHNSON, SKYLAR - Washington State University
item RICE, JENNIFER - Washington State University
item SA, NA - Washington State University
item BAUMGARTNER, JOHN - East Carolina University
item PITZER, JOSHUA - East Carolina University
item ROOP, ROY - East Carolina University
item ROJE, SANJA - Washington State University

Submitted to: Journal of Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/12/2022
Publication Date: 8/13/2022
Citation: Yurgel, S., Johnson, S.S., Rice, J., Sa, N., Baumgartner, J., Pitzer, J.E., Roop, R.M., Roje, S. 2022. A novel formamidase is required for riboflavin biosynthesis in invasive bacteria. Journal of Biological Chemistry. 298(9). Article 102377.

Interpretive Summary: Although bacteria are typically thought of as causes of disease, there are actually many beneficial bacteria that contribute to crop production. For example, roots of legumes including alfalfa, clover, chickpea, and peas are colonized by beneficial bacteria that convert atmospheric nitrogen to nitrogen fertilizer for the legume. The beneficial bacteria that produce nitrogen fertilizer in the roots of legumes are generally referred to as 'rhizobacteria'. The bacteria produce riboflavin, which is essential for several different processes, including replication and repair of bacterial DNA and the ability of rhizobacteria to colonize and produce nitrogen fertilizer in plant roots. The objective of this research was to study the genetic control of riboflavin production by the bacteria Sinorhizobium meliloti, which colonizes alfalfa. This bacteria both secretes riboflavin-like compounds into the external environment and also uses these compounds to support its growth. We determined that the ribBA gene was primarily involved in producing riboflavin-like compounds that are secreted. We also identified a new enzyme (ArfB) involved in the production of riboflavin for growth functions in S. meliloti. We demonstrated that related enzymes from the human pathogen Brucella abortus and the plant disease causing pathogen Liberobacter solanacearum could function in the beneficial rhizobacteria S. meliloti. Our results are significant because improved understanding of riboflavin biosynthesis in bacteria contributes to resolving challenges to agriculture and medicine associated with bacterial infections.

Technical Abstract: 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 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 the product of the ribBA gene participates in the biosynthesis of flavins destined for secretion, and that S. meliloti has another enzyme that performs this function for the needs of the internal cellular metabolism. In this study, 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 SmArfB, 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 bacterial, are highly abundant in the Rhizobiales order, and that the sequence homologs from Brucella abortus and Liberobacter solanacearum complement the riboflavin auxotrophy of the Sm1021'SMc02977 mutant. Furthermore, we show that the B. abortus enzyme (Bab2_0247, BaArfB) 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. In addition, we show that SmArfB and BaArfB physically interact with other enzymes on the riboflavin biosynthesis pathway. The presented findings are significant because they show that riboflavin biosynthesis in bacteria is less understood than previously thought, that different modules synthesize riboflavin for secretion and internal needs in many species of bacteria. Improved understanding of riboflavin biosynthesis in these species is therefore expected to aid agriculture and medicine by providing methods that respectively help establish more efficiently the symbioses between plants and nitrogen-fixing bacteria and help reduce virulence of some bacterial pathogens that infect humans and livestock.