Phosphorylation and Functional Properties of the IIA Domain of the Lactose Transport Protein of Streptococcus thermophilus

Abstract

ABSTRACT The lactose-H + symport protein (LacS) of Streptococcus thermophilus has a carboxyl-terminal regulatory domain (IIA LacS ) that is homologous to a family of proteins and protein domains of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) in various organisms, of which IIA Glc of Escherichia coli is the best-characterized member. On the basis of these similarities, it was anticipated that IIA LacS would be able to perform one or more functions associated with IIA Glc , i.e., carry out phosphoryl transfer and/or affect other catabolic functions. The gene fragment encoding IIA LacS was overexpressed in Escherichia coli , and the protein was purified in two steps by metal affinity and anion-exchange chromatography. IIA LacS was unable to restore glucose uptake in a IIA Glc -deficient strain, which is consistent with a very low rate of phosphorylation of IIA LacS by phosphorylated HPr (HPr∼P) from E. coli . With HPr∼P from S. thermophilus , the rate was more than 10-fold higher, but the rate constants for the phosphorylation of IIA LacS ( k 1 = 4.3 × 10 2 M −1 s −1 ) and dephosphorylation of IIA LacS ∼P by HPr ( k −1 = 1.1 × 10 3 M −1 s −1 ) are still at least 4 orders of magnitude lower than for the phosphoryltransfer between IIA Glc and HPr from E. coli . This finding suggests that IIA LacS has evolved into a protein domain whose main function is not to transfer phosphoryl groups rapidly. On the basis of sequence alignment of IIA proteins with and without putative phosphoryl transfer functions and the known structure of IIA Glc , we constructed a double mutant [IIA LacS (I548E/G556D)] that was predicted to have increased phosphoryl transfer activity. Indeed, the phosphorylation rate of IIA LacS (I548E/G556D) by HPr∼P increased ( k 1 = 4.0 × 10 3 M −1 s −1 ) and became nearly independent of the source of HPr∼P ( S. thermophilus , Bacillus subtilis , or E. coli ). The increased phosphoryl transfer rate of IIA LacS (I548E/G556D) was insufficient to complement IIA Glc in PTS-mediated glucose transport in E. coli . Both IIA LacS and IIA LacS (I548E/G556D) could replace IIA Glc , but in another function: they inhibited glycerol kinase (inducer exclusion) when present in the unphosphorylated form.