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

Abstract

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 PLBPB 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, in which mutant variant PipYK26A but not PipYP63L or PipYR210Q would be defective. A model integrating our observations with previous data from other organims and PLPBP variants is discussed.