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The phosphotransferase system (PTS) of Streptomyces coelicolor identification and biochemical analysis of a histidine phosphocarrier protein HPr encoded by the gene ptsH.
- Source :
-
European journal of biochemistry [Eur J Biochem] 1999 Oct 01; Vol. 265 (1), pp. 308-17. - Publication Year :
- 1999
-
Abstract
- HPr, the histidine-containing phosphocarrier protein of the bacterial phosphotransferase system (PTS) controls sugar uptake and carbon utilization in low-GC Gram-positive bacteria and in Gram-negative bacteria. We have purified HPr from Streptomyces coelicolor cell extracts. The N-terminal sequence matched the product of an S. coelicolor orf, designated ptsH, sequenced as part of the S. coelicolor genome sequencing project. The ptsH gene appears to form a monocistronic operon. Determination of the evolutionary relationship revealed that S. coelicolor HPr is equally distant to all known HPr and HPr-like proteins. The presumptive phosphorylation site around histidine 15 is perfectly conserved while a second possible phosphorylation site at serine 47 is not well-conserved. HPr was overproduced in Escherichia coli in its native form and as a histidine-tagged fusion protein. Histidine-tagged HPr was purified to homogeneity. HPr was phosphorylated by its own enzyme I (EI) and heterologously phosphorylated by EI of Bacillus subtilis and Staphylococcus aureus, respectively. This phosphoenolpyruvate-dependent phosphorylation was absent in an HPr mutant in which histidine 15 was replaced by alanine. Reconstitution of the fructose-specific PTS demonstrated that HPr could efficiently phosphorylate enzyme IIFructose. HPr-P could also phosphorylate enzyme IIGlucose of B. subtilis, enzyme IILactose of S. aureus, and IIAMannitol of E. coli. ATP-dependent phosphorylation was detected with HPr kinase/phosphatase of B. subtilis. These results present the first identification of a gene of the PTS complement of S. coelicolor, providing the basis to elucidate the role(s) of HPr and the PTS in this class of bacteria.
- Subjects :
- Adenosine Triphosphate metabolism
Amino Acid Sequence
Bacillus subtilis genetics
Cloning, Molecular
Consensus Sequence
Escherichia coli
Escherichia coli Proteins
Evolution, Molecular
Genetic Complementation Test
Molecular Sequence Data
Monosaccharide Transport Proteins
Peptide Fragments chemistry
Phosphoenolpyruvate Sugar Phosphotransferase System classification
Phosphoenolpyruvate Sugar Phosphotransferase System metabolism
Phosphorylation
Phosphotransferases (Nitrogenous Group Acceptor) metabolism
Phylogeny
Protein Serine-Threonine Kinases metabolism
Recombinant Proteins metabolism
Sequence Analysis, Protein
Sequence Homology, Amino Acid
Staphylococcus aureus genetics
Bacterial Proteins
Genes, Bacterial
Phosphoenolpyruvate Sugar Phosphotransferase System genetics
Streptomyces genetics
Subjects
Details
- Language :
- English
- ISSN :
- 0014-2956
- Volume :
- 265
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- European journal of biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 10491187
- Full Text :
- https://doi.org/10.1046/j.1432-1327.1999.00727.x