Mutational analyses reveal PLP-independent functions at PipY, the cyanobacterial paradigm for pyridoxal-phosphate binding proteins

Pyridoxal-phosphate binding proteins (PLPBP) are involved in the homeostasis of B₆ vitamers and amino/keto acids, share a high degree of sequence conservation and are represented in all three domains of life. Despite the obligate presence of the catalyst cofactor PLP, attempts to show enzymatic activity have been unsuccessful. Instead, evidence of RNA binding activity has been provided for several members of the family. Here we use PipY, one of the few PLPBP members studied so far, as a model system to address the phenotypic impact in the cyanobacterium Synechococcus elongatus of mutations K26A, P63L and R210Q, which respectively prevent PLP binding or are equivalent to those conferring B₆-dependent epilepsy in humans with a recessive inheritance pattern. We found that while mutation K26A at the PLP-binding residue abrogated all phenotypes associated to PipY overexpression and toxicity, P63L and R210Q behaved as dominant gain-of-function mutations that inhibited bacterial growth. We provide in vivo evidence of PipY performing PLP-independent functions, for which mutant variant PipY^K26A but not PipY^P63L or PipY^R210Q would be defective. A model integrating our observations with previous data from other organisms and PLPBP variants is discussed.