Genetic and biochemical properties of an alkaline phosphatase PhoX family protein found in many bacteria.
We report on the biochemical, phylogenetic and genetic regulation of PhoX, the major alkaline phosphatase protein from the soil bacterium Sinorhizobium meliloti. The protein is shown to be a member of a recently identified family of PhoX alkaline phosphatase proteins that is distinct from the well-characterized PhoA family. The mature S. meliloti PhoX protein is located in the periplasm and lacks a 76-amino-acid N-terminal Tat signal peptide. Its phosphatase activity was stimulated by Ca(+2) and was optimal at pH 9-11. Except for phytic acid and phosphatidic acid, the enzyme was active against a wide range of phosphorylated substrates (77 nucleotides, phosphorylated carbohydrates and amino acids) and thus exhibited low substrate specificity for C-O-P bonds. No C-P bond substrate was dephosphorylated while the protein was active with two of six phosphoramidate substrates (N-P bond) tested. Sinorhizobium meliloti phoX was induced when cells were starved for phosphorous and the induction was dependent on the PhoB-regulatory protein. We demonstrate by in vitro analysis that PhoB protein binds to two tandem 22 nt PhoB binding sites located 64-21 nt upstream from the phoX transcription start site. Analysis of 95 PhoX orthologues from diverse bacteria revealed two distinct phylogenetic groups of PhoX proteins. The two groups differed in having a conserved glycine (PhoX-I) or asparagine (PhoX-II) next to their putative catalytic Ca(+2) binding site. Analysis of the phoX promoter regions from many of these bacteria also revealed the presence of PhoB binding sites. Alkaline phosphatase proteins of either the PhoX or PhoA family (but rarely both) are found in many bacteria, thus it appears that these are functionally equivalent.
Zaheer
Rahat
R
Center for Environmental Genomics, Department of Biology, McMaster University, Hamilton, Ontario, Canada.
Morton
Richard
R
Proudfoot
Mike
M
Yakunin
Alexander
A
Finan
Turlough M
TM
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001419:Bacteria / Q000145:classification; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D015696:Gene Products, tat / Q000737:chemistry; D007477:Ions; D008969:Molecular Sequence Data; D010802:Phylogeny; D016962:Sinorhizobium meliloti / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D018797:Cell Cycle Proteins; D015696:Gene Products, tat; D007477:Ions; C118650:SMC protein, Bacteria; C061278:PhoB protein, Bacteria; D000469:Alkaline Phosphatase; D002118:Calcium