The lactose transport protein (LacS) of Streptococcus thermophilus is a chimeric protein consisting of an amino-terminal carrier domain and a carboxyl-terminal phosphoenolpyruvate:sugar phosphotransferase system (PTS) IIA protein domain. The histidine residues of LacS were changed individually into glutamine or arginine residues. Of the 11 histidine residues present in LacS, only the His-376 substitution in the carrier domain significantly affected sugar transport. The region around His-376 was found to exhibit sequence similarity to the region around His-322 of the lactose transport protein (LacY) of Escherichia coli, which has been implicated in sugar binding and in coupling of sugar and H+ transport. The H376Q mutation resulted in a reduced rate of uptake and altered affinity for lactose (beta-galactoside), melibiose (alpha-galactoside), and the lactose analog methyl-beta-D-thiogalactopyranoside. Similarly, the extent of accumulation of the galactosides by cells expressing LacS(H376Q) was highly reduced in comparison to cells bearing the wild-type protein. Nonequilibrium exchange of lactose and methyl-beta-D-thiogalactopyranoside by the H376Q mutant was approximately 2-fold reduced in comparison to the activity of the wild-type transport protein. The data indicate that His-376 is involved in sugar recognition and is important, but not essential, for the cotransport of protons and galactosides.
The carboxyl-terminal domain of LacS contains 2 histidine residues (His-537 and His-552) that are conserved in seven homologous IIA protein(s) (domains) of PTSs. P-enolpyruvate-dependent phosphorylation of wild-type LacS, but not of the mutant H552Q, was demonstrated using purified Enzyme I and HPr, the general energy coupling proteins of the PTS, and inside-out membrane vesicles isolated from E. coli in which the lactose transport gene was expressed. The His-537 and His-552 mutations did not affect transport activity when the corresponding genes were expressed in E. coli.
|Number of pages||8|
|Journal||The Journal of Biological Chemistry|
|Publication status||Published - 5-May-1992|
- SUGAR PHOSPHOTRANSFERASE SYSTEM
- DEPENDENT PROTON TRANSPORT
- SITE-DIRECTED MUTANTS
- LAC PERMEASE
- MELIBIOSE CARRIER
- ENHANCED RECOGNITION