Alkaline phosphatase from the hyperthermophilic bacterium T. maritima requires cobalt for activity.
The hyperthermophilic bacterium Thermotoga maritima encodes a gene sharing sequence similarities with several known genes for alkaline phosphatase (AP). The putative gene was isolated and the corresponding protein expressed in Escherichia coli, with and without a predicted signal sequence. The recombinant protein showed phosphatase activity toward the substrate p-nitrophenyl-phosphate with a k(cat) of 16 s(-1) and a K(m) of 175 microM at a pH optimum of 8.0 when assayed at 25 degrees C. T. maritima phosphatase activity increased at high temperatures, reaching a maximum k(cat) of 100 s(-1), with a K(m) of 93 microM at 65 degrees C. Activity was stable at 65 degrees C for >24 h and at 90 degrees C for 5 h. Phosphatase activity was dependent on divalent metal ions, specifically Co(II) and Mg(II). Circular dichroism spectra showed that the enzyme gains secondary structure on addition of these metals. Zinc, the most common divalent metal ion required for activity in known APs, was shown to inhibit the T. maritima phosphatase enzyme at concentrations above 0.3 moles Zn: 1 mole monomer. All activity was abolished in the presence of 0.1 mM EDTA. The T. maritima AP primary sequence is 28% identical when compared with E. coli AP. Based on a structural model, the active sites are superimposable except for two residues near the E. coli AP Mg binding site, D153 and K328 (E. coli numbering) corresponding to histidine and tryptophan in T. maritima AP, respectively. Sucrose-density gradient sedimentation experiments showed that the protein exists in several quaternary forms predominated by an octamer.
Wojciechowski
Cheryl L
CL
Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02467, USA.
Cardia
James P
JP
Kantrowitz
Evan R
ER
D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D003035:Cobalt / Q000737:chemistry*; D017931:DNA Primers / Q000737:chemistry; D019281:Dimerization; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics
D017931:DNA Primers; D003035:Cobalt; D000469:Alkaline Phosphatase