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 coil sensory kinase, ArcB, possesses a histidine-conta ining phosphotransfer (HPt) domain, which is implicated in the His-Asp multistep phosphorelay. We searched for a presumed phosphohistidine p hosphatase, if present, which affects the function of the HPt domain t hrough its dephosphorylation activity. Using in vivo screening, we fir st identified a gene that appeared to interfere with the His-Asp phosp horelay between the HPt domain and the receiver domain of OmpR, provid ed that the gene product was expressed through a multicopy plasmid. Th e cloned gene (named sixA) was found to encode a protein consisting of 161 amino acids, which has a noticeable sequence motif, an arginine-h istidine-glycine (RHG) signature, at its N-terminus. Such an RHG signa ture, which presumably functions as a nucleophilic phosphoacceptor, wa s previously found in a set of divergent enzymes, including eukaryotic fructose-2,6-bisphosphatase, E. coil periplasmic phosphatase and E. c oil glucose-l-phosphate phosphatase, and ubiquitous phosphoglycerate m utase. Otherwise, the entire amino acid sequences of none of these enz ymes resembles that of SixA. It was demonstrated in vitro that the pur ified SixA protein exhibited the ability to release the phosphoryl gro up from the HPt domain of ArcB, but the mutant protein lacking the cru cial 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.