An Escherichia coli protein that exhibits phosphohistidine phosphatase activity towards the HPt domain of the ArcB sensor involved in the multistep His-Asp phosphorelay.
The Escherichia coli sensory kinase, ArcB, possesses a histidine-containing phosphotransfer (HPt) domain, which is implicated in the His-Asp multistep phosphorelay. We searched for a presumed phosphohistidine phosphatase, if present, which affects the function of the HPt domain through its dephosphorylation activity. Using in vivo screening, we first identified a gene that appeared to interfere with the His-Asp phosphorelay between the HPt domain and the receiver domain of OmpR, provided that the gene product was expressed through a multicopy plasmid. The cloned gene (named sixA) was found to encode a protein consisting of 161 amino acids, which has a noticeable sequence motif, an arginine-histidine-glycine (RHG) signature, at its N-terminus. Such an RHG signature, which presumably functions as a nucleophilic phosphoacceptor, was previously found in a set of divergent enzymes, including eukaryotic fructose-2,6-bisphosphatase, E. coli periplasmic phosphatase and E. coli glucose-1-phosphate phosphatase, and ubiquitous phosphoglycerate mutase. Otherwise, the entire amino acid sequences of none of these enzymes resembles that of SixA. It was demonstrated in vitro that the purified SixA protein exhibited the ability to release the phosphoryl group from the HPt domain of ArcB, but the mutant protein lacking the crucial histidine residue in the RHG signature did not. Evidence was also provided that a deletion mutation of sixA on the chromosome affected the in vivo phosphotransfer signalling. These results support the view that SixA is capable of functioning as a phosphohistidine phosphatase that may be implicated in the His-Asp phosphorelay through regulating the phosphorylation state of the HPt domain.
Ogino
T
T
Laboratory of Molecular Microbiology, School of Agriculture, Nagoya University, Japan.
Matsubara
M
M
Kato
N
N
Nakamura
Y
Y
Mizuno
T
T
D000595:Amino Acid Sequence; D019677:Artificial Gene Fusion; D001224:Aspartic Acid / Q000378:metabolism; D001345:Autoradiography; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D029968:Escherichia coli Proteins; D017353:Gene Deletion; D005798:Genes, Bacterial / Q000235:genetics; D006639:Histidine / Q000031:analogs & derivatives / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000378:metabolism; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011494:Protein Kinases; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014170:Transformation, Genetic; D001616:beta-Galactosidase
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D010710:Phosphates; D001224:Aspartic Acid; D006639:Histidine; D011494:Protein Kinases; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022409:protein-N-pi-phosphohistidine - sugar phosphotransferase; C065271:arcB protein, E coli; D001616:beta-Galactosidase; C005436:phosphohistidine