BIOCHEMICAL-CHARACTERIZATION OF WRBA, FOUNDING MEMBER OF A NEW FAMILYOF MULTIMERIC FLAVODOXIN-LIKE PROTEINS

The protein WrbA had been identified as an Escherichia coli stationary -phase protein that copurified and coimmunoprecipitated with the trypt ophan repressor. Sequences homologous to WrbA have been reported in se veral species of yeast and plants. We previously showed that this new family of proteins displays low but structurally significant sequence similarity with flavodoxins and that its members are predicted to shar e the alpha/beta core of the flavodoxin fold but with a short conserve d insertion unique to the new family, which could account for reports that some family members may be dimeric in solution. The general seque nce similarity to flavodoxins suggests that the members of the new fam ily might bind FMN, but their wide evolutionary distribution indicates that, unlike the flavodoxins, these proteins may be ubiquitous. In th is paper, we report the purification and biochemical characterization of WrbA, demonstrating that the protein binds FMN specifically and is a multimer in solution. The FMN binding constant is weaker than for ma ny flavodoxins, being similar to 2 mu M at 25 degrees C in 0.1 mM sodi um phosphate, pH 7.2, The protein participates in a dimer-tetramer equ ilibrium over a wide range of solution conditions, with a midpoint at approximately 1.4 mu M. One FMN binds per monomer and has no apparent effect on the multimerization equilibrium. WrbA has no effect on the a ffinity or mode of DNA binding by the tryptophan repressor; thus, its physiological role remains unclear. Although many proteins with flavod oxin-like domains are known to be multimers, WrbA is apparently the fi rst characterized case in which multimerization is associated directly with the flavodoxin-like domain itself.