Synthesis of HPr(Ser-P)(His-P) by enzyme I of the phosphoenolpyruvate: sugar phosphotransferase system of Streptococcus salivarius.
HPr is a protein of the bacterial phosphoenolpyruvate:sugar phosphotransferase transport system (PTS). In Gram-positive bacteria, HPr can be phosphorylated on Ser(46) by HPr(Ser) kinase/phosphorylase (HPrK/P) and on His(15) by enzyme I (EI) of the PTS. In vitro studies have shown that phosphorylation on one residue greatly inhibits the second phosphorylation. However, streptococci contain significant amounts of HPr(Ser-P)(His approximately P) during exponential growth, and recent studies suggest that phosphorylation of HPr(Ser-P) by EI is involved in the recycling of HPr(Ser-P)(His approximately P). We report in this paper a study on the phosphorylation of Streptococcus salivarius HPr, HPr(Ser-P), and HPr(S46D) by EI. Our results indicate that (i) the specificity constant (k(cat)/K(m)) of EI for HPr(Ser-P) at pH 7.9 was approximately 5000-fold smaller than that observed for HPr, (ii) no metabolic intermediates were able to stimulate HPr(Ser-P) phosphorylation, (iii) the rate of HPr phosphorylation decreased at pHs below 6.5, while that of HPr(Ser-P) increased and was almost 10-fold higher at pH 6.1 than at pH 7.9, (iv) HPr(S46D), a mutated HPr alleged to mimic HPr(Ser-P), was also phosphorylated more efficiently under acidic conditions, and, lastly, (v) phosphorylation of Bacillus subtilis HPr(Ser-P) by B. subtilis EI was also stimulated at acidic pH. Our results suggest that the high levels of HPr(Ser-P)(His approximately P) in streptococci result from the combination of two factors, a high physiological concentration of HPr(Ser-P) and stimulation of HPr(Ser-P) phosphorylation by EI at acidic pH, an intracellular condition that occurs in response to the acidification of the external medium during growth of the culture.
Casabon
Israël
I
Groupe de Recherche en Ecologie Buccale (GREB), Faculté de Médecine Dentaire, and Département de Biochimie et de Microbiologie, Faculté des Sciences et de Génie, Université Laval, Quebec City, Quebec, Canada G1K 7P4.
Couture
Manon
M
Vaillancourt
Katy
K
Vadeboncoeur
Christian
C
D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D006868:Hydrolysis; D007700:Kinetics; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013291:Streptococcus / Q000201:enzymology*
D017931:DNA Primers; D011994:Recombinant Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System