Phosphorylation and functional properties of the IIA domain of the lactose transport protein of Streptococcus thermophilus

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(LacC) 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 similar to P) from E. coli. With HPr similar to 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 x 102 M-1 s(-1)) and dephosphorylation of IIA(LacS)similar to P by HPr (k(-1) = 1.1 x 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/G55GD) by HPr similar to P increased (k(1) = 4.0 x 10(3) M-1 s(-1)) and became nearly independent of the source of HPr similar to P (S. thermophilus, Bacillus subtilis, or E. coli). The increased phosphoryl transfer rate of IIA(LacS) (I548E/C556D) 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.