Alkaline phosphatase from the hyperthermophilic bacterium T. maritima requires cobalt for activity

Protein Science : a Publication of the Protein Society
Cheryl L WojciechowskiE R Kantrowitz

Abstract

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 o...Continue Reading

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Citations

Jul 11, 2012·Biotechnology Letters·Kosuke MinamihataNoriho Kamiya
Aug 28, 2003·Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology·Bjarni AsgeirssonPeter Højrup
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Mar 29, 2008·Protein Science : a Publication of the Protein Society·Clare J McClevertyScott A Lesley
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Oct 26, 2002·The Journal of Biological Chemistry·Cheryl L Wojciechowski, Evan R Kantrowitz
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