Enzymatic and Mutational Analysis of the PruA Pteridine Reductase Required for Pterin-Dependent Control of Biofilm Formation in Agrobacterium tumefaciens

Biofilms are complex multicellular communities that are formed by diverse bacteria. In the plant pathogen Agrobacterium tumefaciens, the transition from a free-living motile state to a nonmotile biofilm state is governed by a novel signaling pathway involving small molecules called pterins. The involvement of pterins in biofilm formation is unexpected and prompts many questions about the molecular details of this pathway. This work biochemically characterized the PruA pteridine reductase involved in the signaling pathway to reveal its enzymatic properties and substrate preference, thus providing important insight into pterin biosynthesis and its role in A. tumefaciens biofilm control. Additionally, the enzymatic characteristics of related pteridine reductases from mammalian pathogens were examined to uncover potential roles of these enzymes in other bacteria. ABSTRACT Pterins are ubiquitous biomolecules with diverse functions, including roles as cofactors, pigments, and redox mediators. Recently, a novel pterin-dependent signaling pathway that controls biofilm formation was identified in the plant pathogen Agrobacterium tumefaciens. A key player in this pathway is a pteridine reductase, termed PruA, where its enzymatic activity has been shown to control surface attachment and limit biofilm formation. Here, we biochemically characterized PruA to investigate the catalytic properties and the substrate specificity of this pteridine reductase. PruA demonstrated maximal catalytic efficiency with dihydrobiopterin and comparable activities with the stereoisomers dihydromonapterin and dihydroneopterin. Since A. tumefaciens does not synthesize or utilize biopterins, the likely physiological substrate is dihydromonapterin or dihydroneopterin or both. Notably, PruA did not exhibit pteridine reductase activity with dihydrofolate or fully oxidized pterins. Site-directed mutagenesis studies of a conserved tyrosine residue, the key component of a putative catalytic triad, indicated that this tyrosine is not directly involved in PruA catalysis but may be important for substrate or cofactor binding. Additionally, mutagenesis of the arginine residue in the N-terminal TGX3RXG motif significantly reduced the catalytic efficiency of PruA, supporting its proposed role in pterin binding and catalysis. Finally, we report on the enzymatic characterization of PruA homologs from Pseudomonas aeruginosa and Brucella abortus, thus expanding the roles and potential significance of pteridine reductases in diverse bacteria. IMPORTANCE Biofilms are complex multicellular communities that are formed by diverse bacteria. In the plant pathogen Agrobacterium tumefaciens, the transition from a free-living motile state to a nonmotile biofilm state is governed by a novel signaling pathway involving small molecules called pterins. The involvement of pterins in biofilm formation is unexpected and prompts many questions about the molecular details of this pathway. This work biochemically characterized the PruA pteridine reductase involved in the signaling pathway to reveal its enzymatic properties and substrate preference, thus providing important insight into pterin biosynthesis and its role in A. tumefaciens biofilm control. Additionally, the enzymatic characteristics of related pteridine reductases from mammalian pathogens were examined to uncover potential roles of these enzymes in other bacteria.