Phosphorylation of mycobacterial phosphodiesterase by eukaryotic-type Ser/Thr kinase controls its two distinct and mutually exclusive functionalities.

Phosphorylation-mediated negative feedback regulation of cAMP levels by phosphodiesterase is well-established in eukaryotic cells. However, such a mechanism remains unexplored in prokaryotes. We report here the involvement of eukaryotic-type Ser/Thr kinases, particularly PknA in trans-phosphorylating phosphodiesterase from (mPDE), that resulted in decreased enzyme turnover rate compared with its unphosphorylated counterpart. To elucidate the role of mPDE phosphorylation in hydrolyzing cellular cAMP, we utilized a phosphodiesterase knock-out strain, Δ, where interference of endogenous eukaryotic-type Ser/Thr kinases could be excluded. Interestingly, the mPDE-complemented Δ strain showed enhanced cAMP levels in the presence of PknA, and this effect was antagonized by PknA-K42N, a kinase-dead variant. Structural analysis of mPDE revealed that four Ser/Thr residues (Ser-20, Thr-22, Thr-182, and Thr-240) were close to the active site, indicating their possible role in phosphorylation-mediated alteration in enzymatic activity. Mutation of these residues one at a time to alanine or a combination of all four (mPDE-4A) affected catalytic activity of mPDE. Moreover, mPDE-4A protein in kinase assays exhibited reduction in its phosphorylation compared with mPDE. In consonance, phosphoproteins obtained after co-expression of PknA with mPDE/S20A/T240A/4A displayed decreased phospho-signal intensities in immunoblotting with anti-phosphoserine/phosphothreonine antibodies. Furthermore, unlike mPDE, phospho-ablated mPDE-T309A protein exhibited impaired cell wall localization in , whereas mPDE-4A behaved similarly as wild type. Taken together, our findings establish mutually exclusive dual functionality of mPDE upon PknA-mediated phosphorylation, where Ser-20/Thr-240 influence enzyme activity and Thr-309 endorses its cell wall localization.